Dissertation Osteopathy

Research Project

Effects of indirect osteopathic techniques on heart rate variability -nonlinear analyses included- and on electrodermal activity in healthy adults.

The RILHOU Randomized Clinical Trial.

Effects of a set of cranial techniques on the autonomic nervous system in a cohort of healthy adults.

Scientific paper to acquire the title ‘D. O. IN OSTEOPATHY’ granted by the FICO September, 2017 Made and presented by:

Wens Chris (Contact person RILHOU-RCT)

Winkelhaakstraat 28, 2170 Merksem, Belgium Study coordinator:

Smeets Hubert, D. O.

Scientific coordinator:

De Cock Johan, PhD, MSc, D. O. (†)

Coordinator dissertations FICO: Quaghebeur Jörgen, PhD, MSc, D. O.

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Author: Wens Chris Winkelhaakstraat 28, 2170 Merksem Belgium wenschris@hotmail. Com

+32(0)471793411

Flanders International College of Osteopathy Student ID: 2007/ 18

Declaration

I hereby assure that the presented dissertation is compiled by my own hands and has not been submitted elsewhere for examination/review. Foreign sources are identified and labelled with information about their origin. D/2017/Wens Chris Alfons Ria, author.

Wens Chris Merksem, 21/05/2017 (Contact person RILHOU-RCT)

Study coordinator:

Scientific coordinator:

Smeets Hubert, D. O.

De Cock Johan, PhD, MSc, D. O. (†6 December 2016)

Coordinator dissertations FICO: Quaghebeur JĂśrgen, PhD, MSc, D. O.

1

Abstract NAME:

Wens

Chris

STUDY

COORDINATOR:

Smeets

Hubert

SCIENTIFIC

COORDINATOR: De Cock Johan, COORDINATOR FICO: Quaghebeur Jörgen. Year: 2017 TITLE: Effects of indirect osteopathic techniques on heart rate variability nonlinear analyses included- and on electrodermal activity in healthy adults. The RILHOU Randomized Clinical Trial (RCT). RESEARCH TITLE: Effects of a set of cranial techniques on the autonomic nervous system in a cohort of healthy adults.

BACKGROUND: The autonomic nervous system (ANS) supplies every organ and tissue of the body. ANS imbalance is an underlying mechanism for a various of psychophysiological disorders. The parameters below are acceptable for measure ANS activity. OBJECTIVE: To investigate the potential effects of a set of cranial techniques on the ANS. METHOD: The RILHOU Randomized Clinical Trial (RCT), Eudract/B-nr: B371201422023 is a sham controlled RCT (according to the ICH-GCP guidelines, subordinate to a Failure Modes and Effects analysis) investigate the direct and shortterm (treatment) effects of a set of cranial techniques on: heart rate variability (HRV), skin conduction level (SCL), respiratory sinus arrhythmia (RSA), pre-ejection period (PEP) and blood pressure (BP). The methodology of the study was standardized and described in Common Documents, public accessible. BP was measured with Omron.

HRV, SCL, RSA and PEP were obtained with the VU-AMS device 5fs. VU-DAMS v3.9 and Kubios v2.2 were used to analyse HRV. Data from different time periods were analyzed with the Wilcoxon Ranks Test (WRT) and ANCOVA (A). RESULTS: Forty healthy subjects (31.45 ± 6.47 years of age; 23.05 ± 2.81 BMI) showed no significant difference for age or BMI between men and women (Mann-Whitney U Test: p > .05), and were divided into an experimental- and sham group. Only the experimental group showed on short-term an increased (treatment) effect on HRV for: DFA α2 [K] (WRT: p .030, Sham: p .627; A: p < .001), LF/HF ratio FFT [V] (WRT: p .015; A: p .042), LF n.u.

AR [K] (WRT: p .017; A: p .028) with a decrease on HF n.u. AR [K] (WRT: p .017; p .028) and HF ms2 FFT [V] (WRT: p .037; A: p .013). Both groups showed no direct or short-term (treatment) effects for: the other HRV parameters, SCL, RSA, PEP and BP (p < .05 was not found in both tests: WRT, A). DISCUSSION: The experimental- and sham group showed direct an increase for: vagal activity, sympathovagal balance and SCL. PEP and BP were increased in the experimental group, the opposite for the sham. The experimental group showed on short-term a decrease in vagal activity, the opposite for the sham. Both groups reduced PEP and BP, while the level of SCL increased. The used software (VU-DAMS, Kubios) and type of analysis (fast fourier transform or autoregression) gives different conclusions in terms of significance.

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CONCLUSION: Short-term (treatment) effects have been shown in the experimental group on HRV with frequency domain and nonlinear analysis.

5 Keywords: osteopathy, ICH-GCP, FMEA, HRV, VU-AMS.

3

Abstract NAAM:

Wens

Chris

STUDIE

COÖRDINATOR:

Smeets

Hubert

WETENSCHAPPELIJKE COÖRDINATOR: De Cock Johan, COÖRDINATOR FICO: Quaghebeur Jörgen. JAAR: 2017 TITEL: Effecten van indirecte osteopathische technieken op hartslagvariabiliteit -niet lineaire analyses- en op electrodermale activiteit bij

gezonde

volwassenen.

De

RILHOU

gerandomiseerde

klinische

studie.

ONDERZOEKSTITEL: Effecten van een set craniale technieken op het autonome zenuwstelsel in een cohort van gezonde volwassenen. ACHTERGROND: Het autonome zenuwstelsel (ANS) regelt elk orgaan en weefsel in het lichaam. ANS imbalans is een onderliggend mechanisme voor diverse psycho-fysiologische stoornissen. Onderstaande parameters zijn aanvaarbaar voor het meten van ANS activiteit. DOEL: Het effect nagaan van een set craniale technieken op het ANS. METHODOLOGIE: De RILHOU Randomized Clinical Trial (RCT), Eudract/B-nr: B371201422023 is een sham gecontroleerde RCT (overeenkomstig met de ICH-GCP richtlijnen, onderworpen aan een Faal Modus en Effecten analyse) onderzoekt het onmiddelijk en korte termijn (behandelings) effect van een set craniale technieken op: hartslagvariabiliteit (HRV), huidgeleiding niveau (SCL), respiratoire sinus aritmie (RSA), pre-ejectie

periode

(PEP)

en

bloeddruk

(BP).

De

methodologie

werd

gestandaardiseerd en beschreven in Common Documents, publiek toegankelijk.

Bloeddruk werd gemeten met Omron. HRV, SCL, RSA en PEP werd bekomen via het VU-AMS 5fs apparaat. VU-DAMS v3.9 en Kubios v2.2 werden gebruikt om HRV te analyseren. Data van verschillende tijdsperiodes werden geanalyseerd via de Wilcoxon Ranks Test (WRT) en ANCOVA (A). RESULTATEN: Veertig gezonde proefpersonen (31.45 ± 6.47 leeftijd; 23.05 ± 2.81 BMI) vertoonden geen significant verschil tussen mannen en vrouwen voor leeftijd en BMI (Mann-Whitney U Test: p > .05), en werden onderverdeeld in een experimentele- en sham groep. Enkel de experimentele groep vertoonde op korte termijn een verhoogd (behandelings) effect op HRV voor: DFA α2 [K] (WRT: p .030, Sham: p .627; A: p < .001), LF/HF ratio FFT [V] (WRT: p .015; A: p .042), LF n.u. AR [K] (WRT: p .017; A: p .028) met een verlanging op HF n.u. AR [K] (WRT: p .017; p .028) en HF ms2 FFT [V] (WRT: p .037; A: p .013). Beide groepen vertoonden geen onmiddellijk of korte termijn (behandelings) effecten voor: de overige HRV parameters, SCL, RSA, PEP en BP (p < .05 werd niet tegelijkertijd bevonden in beide testen: WRT, A). DISCUSSIE: De experimentele- en sham groep vertonen onmiddellijk een verhoging op: de vagale activiteit, sympathovagal balans en SCL. PEP en BD was verhoogd in de experimentele groep, het tegenovergestelde van de sham. De experimentele groep vertoont op korte termijn een verlanging op de vagale

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activiteit, het tegengestelde van de sham. Beide groepen reduceren PEP en BP, terwijl het niveau van SCL werd verhoogd. De gebruikte software (VU-DAMS, Kubios) en type van analyse (fast fourier transform of autoregressie) geven verschillende conclusies inzake van significantie. CONCLUSIE: Korte temijn (behandelings) effecten werden bevonden in de experimentele groep voor HRV in frequentiedomein en niet-lineaire analyse.

5 Trefwoorden: osteopathie, ICH-GCP, FMEA, HRV, VU-AMS.

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Contents 1

List of Figures ........................................................................................... 13

2

List of Tables ............................................................................................ 14

3

Introduction............................................................................................... 16

4

Part I: legislation on experiments on the human person............................ 20

4.1

Historical review ....................................................................................... 20

4.1.1

The Nuremberg Code (1947) .................................................................... 20

4.1.2

Kefauver-Harris Amendment (1962) ......................................................... 21

4.1.3

The Helsinki Declaration (1964) ................................................................ 22

4.1.4

ICH-GCP .................................................................................................. 23

4.2

The Belgian legislative framework concerning experiments on the human person ...................................................................................................... 25

4.2.1

Law of 7 May 2004 concerning experiments on the human person (B. S. 18 May 2004) .................................................................................. 25

4.3

Application of the law of 7 May 2004 ........................................................ 26

4.3.1

Chapter I: General provision ..................................................................... 26

4.3.2

Chapter II: Definitions and application ...................................................... 28

4.3.3

Chapter III: General provision on the protection of participants ................. 34

4.3.4

Chapter IV: Special provision for the protection of minors taking part in experiments .............................................................................................. 41

4.3.5

Chapter V: Special provisions for the protection of adults who are unable to give their consent for participation in experiments ................................ 41

4.3.6

Chapter VI: Special provisions for the protection of persons whose consent cannot be obtained because of urgency ...................................... 42

4.3.7

Chapter VII: Beginning of the experiment ................................................. 42

4.3.8

Chapter VIII: Ethics Committee................................................................. 43

4.3.9

Chapter IX: Admission of the Minister in case of trial ................................ 44

4.3.10

Chapter X: Changes in the implementation of an experiment. .................. 45

4.3.11

Chapter XI: End of the experiment ............................................................ 45

4.3.12

Chapter XII: Suspension or prohibition of the experiment and violations ... 46

4.3.13

Chapter XIV: Review of the agreement with good clinical practice and good manufacturing practice of investigational medicinal products ........... 46

4.3.14

Chapter XV: Reporting of adverse events ................................................. 47

4.3.15

Chapter XVI: Reporting of serious side effects ......................................... 48

4.3.16

Chapter XVII: Liability and insurance ........................................................ 48

4.3.17

Chapter XIX: Special provisions for non-commercial experiments ............ 49

4.3.18

Chapter XX: General ................................................................................ 49

6

4.3.19

Pro memorie ............................................................................................. 50

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Part II: Heart Rate Variability (HRV) .......................................................... 50

5.1

Introduction: the heart ............................................................................... 50

5.2

Communication pathways of the heart ...................................................... 51

5.2.1

Neurological communication ..................................................................... 51

5.2.2

Bio-physiological communication (pulse wave) ......................................... 53

5.2.3

Bio-chemical communication .................................................................... 53

5.2.4

Bio-energetic communication.................................................................... 55

5.3

Heart rate variability .................................................................................. 57

5.3.1

Definition of heart rate variability ............................................................... 58

5.3.2

Background .............................................................................................. 58

5.3.3

General ................................................................................................... 60

6

Part III: The VU-AMS device ..................................................................... 62

6.1

VU-AMS and publications ......................................................................... 63

6.2

The electrocardiogram (ECG) ................................................................... 63

6.3

Impedance cardiography (ICG)................................................................. 64

6.4

Electrodermal activity................................................................................ 67

6.5

VU-AMS measuring variables ................................................................... 68

7

Part IV: similarities and differences between software programme VUDAMS (v3.9) and Kubios (v 2.2) in relation to the HRV parameters .......... 69

7.1

General .................................................................................................... 69

7.1.1

Computational methods ............................................................................ 69

7.1.2

QRST detection algorithm ........................................................................ 70

7.2

Time domain method and geometric analysis ........................................... 71

7.2.1

SDNN

7.2.2

SDANN ................................................................................................... 73

7.2.3

ASDNN ................................................................................................... 73

7.2.4

SD HR ................................................................................................... 73

7.2.5

NN50 and pNN50 ..................................................................................... 73

7.2.6

rMSSD ................................................................................................... 73

7.2.7

Detrended time series............................................................................... 74

7.2.8

RRtri and TINN ......................................................................................... 74

7.3

Frequency domain analysis ...................................................................... 74

7.4

Nonlinear analysis .................................................................................... 77

7.4.1

The Poincaré method ............................................................................... 77

7.4.2

Recurrenc plot .......................................................................................... 78

7.4.3

Entropy ................................................................................................... 78

………………. ............................................................................. 72

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7.4.4

Shannon Entropy ...................................................................................... 78

7.4.5

Approximate Entropy ................................................................................ 78

7.4.6

Sample Entropy ........................................................................................ 78

7.4.7

Detrended Fluctuation Analysis ................................................................ 78

7.4.8

Correlation Dimension Analysis ................................................................ 79

7.5

Respiratory sinus arrhythmia extracted from the ECG .............................. 80

8

Part V: methodology and measurements .................................................. 81

8.1

Measurements .......................................................................................... 81

8.1.1

Research .................................................................................................. 81

8.1.2

Research title ........................................................................................... 82

8.1.3

Research question.................................................................................... 82

8.2

Hypotheses .............................................................................................. 82

8.2.1

Hypothesis related to HRV ....................................................................... 82

8.2.2

Hypothesis relating average SCL ............................................................. 83

8.2.3

Hypothesis related to RSA ........................................................................ 83

8.2.4

Hypothesis related to PEP ........................................................................ 83

8.2.5

Hypothesis related to blood pressure........................................................ 83

8.3

Material and methods ............................................................................... 83

8.3.1

Physiological recording from the VU-AMS 5fs device ............................... 84

8.3.2

Data reduction .......................................................................................... 85

8.3.3

Study type ................................................................................................ 86

8.3.4

Study progress ......................................................................................... 86

8.3.5

Population ................................................................................................ 88

8.3.5.1

Inclusion criteria........................................................................................ 88

8.3.5.2

Exclusion criteria ...................................................................................... 88

8.3.6

Investigation ............................................................................................. 89

8.3.7

Procedure ................................................................................................. 89

8.3.7.1

Randomization ......................................................................................... 89

8.3.7.2

Sham…………………………………… ...................................................... 89

8.3.7.2.1

Technique at the height of the head.......................................................... 89

8.3.7.2.2

Technique at the level of the umbilicus ..................................................... 90

8.3.7.2.3

Technique at the height of the feet ........................................................... 90

8.3.7.3

Experimental techniques .......................................................................... 90

8.3.7.3.1

Techniques experiment according to sequence of execution .................... 90

8.3.7.4

Blinding…… ............................................................................................. 91

8.3.7.5

Processing…. ........................................................................................... 91

8.3.7.5.1

Description of the variables ...................................................................... 91

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8.3.7.5.2

Descriptive statistics ................................................................................. 92

8.3.7.5.3

Normality test ........................................................................................... 93

8.3.7.5.4

The Independent T-test (Student's T-test) ................................................ 93

8.3.7.5.5

The Pearson Chi-square test (point-of-care test) ...................................... 93

8.3.7.5.6

Differences between the same group (treatment variables) on different time periods (Wilcoxon Signed Ranks Test) ..................................................... 94

8.3.7.5.7

The difference between the different groups on different time periods (ANCOVA) ................................................................................................ 95

9

Part VI: Results......................................................................................... 96

9.1

Parametric test for the subject variables (Independent T-test and Pearson Chi-square test). .................................. 97

9.1.1

The difference between the experimental group (R) and the Sham group (S) for the subject variables, scaled ratio (Independent T-test). ................ 97

9.1.2

The difference between the experimental group and the Sham group for the subject variables, scaled nominal (The Pearson Chi-square test). ...... 97

9.2

Non-parametric test for the other variables (Wilcoxon Signed Ranks Test and ANCOVA). ......................................................................................... 98

9.2.1

Summary results Wilcoxon Ranks Test and ANCOVA .............................. 98

9.2.1.1

Comparison baseline (t0) experimental group with Sham ......................... 98

9.2.1.1.1

Total variance of HRV............................................................................... 98

9.2.1.1.2

Parasympathetic activity ........................................................................... 98

9.2.1.1.3

Sympathetic activity .................................................................................. 99

9.2.1.1.4

Sympathovagal balance ......................................................................... 100

9.2.1.1.5

Scaling behaviour of the ANS ................................................................. 100

9.2.1.1.6

Cardiac complexity and regularity ........................................................... 101

9.2.1.1.7

Blood pressure ....................................................................................... 101

9.2.1.2

t1 and t2 compared with the baseline (t0) in the same group (Wilcoxon Ranks Test) and between the different groups (ANCOVA) ..................... 101

9.2.1.2.1

Total variance of HRV............................................................................. 101

9.2.1.2.2

Parasympathetic activity ......................................................................... 103

9.2.1.2.3

Sympathetic activity ................................................................................ 107

9.2.1.2.4

Sympathovagal balance ......................................................................... 108

9.2.1.2.5

Scaling behavior of the ANS ................................................................... 110

9.2.1.2.6

Cardiac complexity and regularity ........................................................... 111

9.2.1.2.7

Blood Pressure ....................................................................................... 112

10

Part VII: Discussion ................................................................................ 114

10.1

Discussion intent of RILHOU-RCT .......................................................... 114

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10.2

Discussion European and Belgian legislation on experiment on the human person. ....................................................................................... 116

10.3

Discussion methodology ......................................................................... 120

10.4

Discussion techniques ............................................................................ 126

10.5

Discussion of the results ......................................................................... 127

10.5.1

Discussion of the results : Comparison baseline (t0)

Experimental group

with Sham............................................................................................... 129 10.5.2

Discussion of the results: t1and t2 compared with the baseline (t0) in the same group (Wilcoxon Ranks Test) and between the different groups (ANCOVA) .................................................................................. 130

10.5.2.1

Total variance of HRV............................................................................. 130

10.5.2.2

Parasympathetic activity ......................................................................... 131

10.5.2.3

Sympathetic activity ................................................................................ 132

10.5.2.4

Sympathovagal balance ......................................................................... 133

10.5.2.5

Scaling behavior of the ANS ................................................................... 134

10.5.2.6

Cardiac complexity and regularity ........................................................... 135

10.5.2.7

Blood pressure ....................................................................................... 136

10.5.3

General................................................................................................... 136

11.

Conclusion.............................................................................................. 138

12.

References ............................................................................................. 139

13.

List of abbreviations ................................................................................ 184

14.

Addenda ................................................................................................. 188

14.1

Addendum 1: Informed consent .............................................................. 189

14.2

Addendum 2: letter of agreement and consent form ............................... 194

14.3

Addendum 3: Flowchart of the four phases at The RILHOU-RCT ........... 196

14.4

Addendum 4: Endocrine heart ................................................................ 197

14.5

Addendum 5: The influence of cardiac hormones on HRV ...................... 199

14.6

Addendum 6: variables that have an impact on various parameters. ...... 202

14.6.1

Relationship age, gender and HRV......................................................... 202

14.6.2

Relation gender and HR ......................................................................... 202

14.6.3

Relationship age and SCL ...................................................................... 202

14.6.4

Relationship sex and SCL ...................................................................... 202

14.6.5

Status and HRV ...................................................................................... 202

14.6.6

Status and BD ........................................................................................ 203

14.6.7

Status and BMI ....................................................................................... 203

14.6.8

Relationship fears and HRV.................................................................... 203

14.6.9

Relationship autoimmune disease and HRV ........................................... 203

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14.6.10

Smoking and HRV Relationship .............................................................. 203

14.6.11

Impact of smoking on blood pressure and heart rate .............................. 204

14.6.12

Impact of smoking on the electro dermal activity..................................... 204

14.6.13

Influence of oral contraception on HRV, BD, RSA .................................. 204

14.6.14

Length, BMI/weight in relationship with HRV .......................................... 204

14.6.15

Relationship BMI with blood pressure and heart rate (BD) ...................... 204

14.6.16

Correlation between BMI, BD, HRV ........................................................ 204

14.6.17

Relation BMI and EDA ............................................................................ 205

14.6.18

Relationship and BMI and body fat% HR ................................................ 205

14.6.19

Ambient temperature and SCL ............................................................... 205

14.6.20

Relationship time and EDA ..................................................................... 205

14.6.21

Relationship HRV day and night ............................................................. 205

14.7

Addendum 7: Hardware VU-AMS 5fs and software VU-AMS, VU-DAMS, Kubios. ................................................................................. 207

14.8

Addendum 8: Socio-demographic data description ................................. 212

14.8.1

Datacollection experiment/Sham en follow-up ........................................ 212

14.8.2

Nationality............................................................................................... 212

14.8.3

Gender….. .............................................................................................. 212

14.8.4

Age……. ................................................................................................. 212

14.8.5

BMI……. ................................................................................................. 212

14.8.6

Civil status .............................................................................................. 213

14.8.7

Number of hours of sports ...................................................................... 213

14.8.8

Shift patterns .......................................................................................... 213

14.8.9

Smoking ................................................................................................. 213

14.8.10

Number of glasses of alcohol a week ..................................................... 213

14.8.11

Allergy… ................................................................................................. 213

14.8.12

Categorical scale related to the experience of pain ................................. 214

14.8.13

Use of non-conventional medicine and/or alternative form ...................... 214

14.8.14

Supplementation of Ω3 fatty acids .......................................................... 214

14.8.15

Medication .............................................................................................. 214

14.8.16

Ambient temprature ................................................................................ 215

14.8.17

Relative humidity percentage.................................................................. 215

14.9

Addendum 9: Measure of central tendency and dispersion: HRV, SCL, RSA, respiration rate, blood pressure ..................................................... 216

14.9.1

HRV: Time domain ................................................................................. 216

14.9.2

HRV: Frequence domain ........................................................................ 219

14.9.3

HRV: Nonlinear analysis ......................................................................... 225

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14.9.4

Other measurements: SCL, PEP, RSA, respiration rate, blood Pressure 229

14.9.5

Regression to the mean.......................................................................... 232

14.10

Addendum 10: Techniques used in the experimental group ................... 233 1. Third Vault Hold .................................................................................. 233 2. Spheno- basilar decompression: ('Platybasia technique') ................... 233 3. J. E. Upledger’s ‘ear pull’ technique .................................................... 233 4. Parietal lift ........................................................................................... 234 5. Frontal lift ............................................................................................ 235 6. Technique for the frontonasal suture .................................................. 235

15

Acknowledgements ................................................................................ 237

12

1 List of Figures Figure 1: Afferent pathways of the nucleus tractus solitarius to the subcortical levels..52 Figure 2: Microscopic image of interconnected intrinsic cardiac ganglia in the human Heart ........................................................................................................... 53 Figure 3: Metabolic communication between cardiokines ANF/ BNP And other tissues ........................................................................................ 54 Figure 4: Effects of cardiokine ANP on the cardiometabolic regulation on blood fibers, Kidneys and adipocytes .............................................................................. 55 Figure 5: Receptor activation by ligands and electromagnetic fields ........................... 56 Figure 6: Synchronization between the brain waves of a mother (EEG) and the ECG Of her baby .......................................................................................................... 56 Figure 7: Wave components PQRST in a normal ECG (U wave is not shown in the figure) ..................................................................... 58 Figure 8: The VU-AMS device 5fs............................................................................... 62 Figure 9: Electrocardiogram with PQRSTU-wave components ................................... 64 Figure 10: Placement of the ECG and ICG electrodes (VU-AMS) ............................... 65 Figure 11: Topography of the skin (eccrine sweat gland) ............................................ 67 Figure 12: Processing block diagram of the ECG to HRV parameters ........................ 71 Figure 13: Relationship between HRV with age and gender ..................................... 206 Figure 14: Possible interrelationships between the different variables ...................... 206

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2 List of Tables Table 1: HRV parameters in different domains ........................................................... 61 Table 2: Impedance-generated hemodynamic parameters and definitions ................. 66 Table 3: Time domain HRV parameters in VU-DAMS v3.9 and Kubios, Used in the RILHOU-RCT ..................................................................................... 72 Table 4: Frequency domain HRV parameters in VU-DAMS v3.9 and Kubios, Used in the RILHOU-RCT ..................................................................................... 76 Table 5: Nonlinear HRV parameters in VU-DAMS v3.9 and Kubios, Used in the RILHOU-RCT ..................................................................................... 79 Table 6: Time scale experiment .................................................................................. 87 Table 7: Time scale follow-up ..................................................................................... 88 Table 8: Follow abbreviations were used for the different variables ............................ 96 Table 9: Independent T-test ........................................................................................ 97 Table 10: Pearson Chi-Square Test ............................................................................ 98 Table 11: Flowchart of four phases of a parallel randomized clinical trial, modified From the Consolidated Standards of Reporting (CONSORT) ............................ 196 Table 12: Substances produced by the vertebrate heart ........................................... 197 Table 13: The influence of cardiac hormones on HRV .............................................. 199 Table 14: Software VU-AMS 5fs. .............................................................................. 207 Table 15: Channels VU-AMS 5fs .............................................................................. 208 Table 16: Measure of central tendency and dispersion for average heart rate [V] ..... 216 Table 17: Measure of central tendency and dispersion for average IBI [V] ................ 217 Table 18: Measure of central tendency and dispersion for rMSSD [V] ...................... 217 Table 19: Measure of central tendency and dispersion for pNN50 [K] ....................... 218 Table 20: Measure of central tendency and dispersion for SDNN [V] ........................ 218 Table 21: Measure of central tendency and dispersion for LF ms2 FFT [V] ............... 219 Table 22: Measure of central tendency and dispersion for LF ms2 AR [K] ................. 219 Table 23: Measure of central tendency and dispersion for LF n.u.FFT [K] ................ 220 Table 24: Measure of central tendency and dispersion for LF n.u. AR [K] ................. 220 Table 25: Measure of central tendency and dispersion for HF ms2 FFT [V]............... 221 Table 26: Measure of central tendency and dispersion for HF ms2 AR [K] ................ 221 Table 27: Measure of central tendency and dispersion for HF n.u.FFT [K] ................ 222 Table 28: Measure of central tendency and dispersion for HF n.u. AR [K] ................ 222 Table 29: Measure of central tendency and dispersion for LF/HF ratio ms2 FFT [K] .. 223 Table 30: Measure of central tendency and dispersion for LF/HF ratio ms2 AR [K] ... 223 Table 31: Measure of central tendency and dispersion for Total power ms2 FFT [K] . 224 Table 32: Measure of central tendency and dispersion for Total power ms2 AR [K] .. 224

14

Table 33: Measure of central tendency and dispersion for ApEn .............................. 225 Table 34: Measure of central tendency and dispersion for SD1 ................................ 225 Table 35: Measure of central tendency and dispersion for SD2 ................................ 226 Table 36: Measure of central tendency and dispersion for D2 .................................. 226 Table 37: Measure of central tendency and dispersion for DFA Îą1 ........................... 227 Table 38: Measure of central tendency and dispersion for DFA Îą2 ........................... 227 Tabel 39: Measure of central tendency and dispersion for Shannon Entropy ............ 228 Table 40: Measure of central tendency and dispersion for SCL ................................ 229 Table 41: Measure of central tendency and dispersion for PEP ................................ 229 Table 42: Measure of central tendency and dispersion for RSA ................................ 230 Table 43: Measure of central tendency and dispersion for respiration rate ............... 230 Table 44: Measure of central tendency and dispersion for SBPR ............................. 231 Table 45: Measure of central tendency and dispersion for DBPR ............................. 231 Table 46: Regression to the mean ............................................................................ 232

15

3 Introduction In Europe, scientific research within osteopathy is largely carried out by students in the framework of thesis projects for earning a Diploma in Osteopathy (D. O.) or Master of Science (MSc) degree in Osteopathy. The demand for Good Clinical Practice (GCP) studies in osteopathy is strongly recommended by the World Health Organization (World Health Organization, 2010; Moore et al. 2014; De Cock, 2014).

From an ethical and legal point of view experiments on the human person are sometimes hard to justify. The subject in an experiment is elevated to a research object, which is not always without risk or danger. These dangers can be reduced by using a risk analysis as a Failure Mode and Effects Analysis (FMEA) (Tarpey et al.

2010; Brennan et al. 2011; Phipps et al. 2011). Possible side effects (positive, negative) in relation to the osteopathic intervention must be recorded and inventoried.

In 2003, in the 'National Council for Osteopathic Research (NCOR)' was founded in the UK. Different projects such as the 'Clinical Risk Osteopathy and Management (CROaM)â&#x20AC;&#x2122; were worked out to ensure safety (risk and benefits), to report possible side effects (adverse events) and to identify the reactions within osteopathy treatment (Vogel et al. 2012, 2013; Vogel, 2010; Carnes et al. 2010a,b). The quest for medical progress is sometimes contrasted with the physical, mental and moral integrity of the test subject in an experiment. The right to respect the moral and physical integrity of patients and volunteers is guaranteed by both international regulations (European Convention on human rights; International Covenant on Civil and political rights (BUPO), as provisions at national level (Belgian Penal Code; Belgian Constitution) (van den Brande and Balthazar, 2010). To provide an answer on the mechanism, the efficiency and effectiveness of the osteopathic intervention a scientific experiment must comply with the Declaration of Helsinki (1964). The guidelines of the International Conference on Harmonization of Technical Requirements for registration of pharmaceuticals for human use and Good Clinical Practice (ICH-GCP) (Kaiser et al.

2013; Schwerla et al. 2013), as well as the legislative framework concerning experiments on the human person of the relevant country are nowadays the key stones in the conduct of any clinical trial. This legal framework for Belgium can be found in the law of 7 May 2004 on experiments, Penal Code, article 392 et seq. and article 12 of the Constitution.

In 2001 a European directive stimulated the EU Member States to develop a law which arranges on a relatively coherent way the legal and ethical aspects of clinical trials

16

(Directive 2001/20/EC). On account of the law of 7 May 2004 concerning trials on human persons (B. S. 18 May 2004) the European directive of 2001 was applied in the Belgian legislation (Dresse, 2005).

This law provides a number of conditions to with a clinical trial must comply before it can start in Belgium (Pinxten et al. 2008). When one is not in accordance with this law and yet performs an experiment on a human person, he commits an infringement on this law, which could have legal implications (B. S. 18 May 2004).

According to the 'Commission for Osteopathic Research Practice and Promotion (CORPP)’ there are two important questions in scientific research related to osteopathy: “Does osteopathy work ?" and "How does it work?".

To be able to give an answer to the first question, it is necessary to take into account the moderators and is formulating statements on the effectiveness of the intervention of interest. The second question can best be answered on the basis of study of the mediators. These can give an explanation model of the mechanism of action of "an intervention", with any statements to the effectiveness of the intervention.

These questions are, according to the CORPP, important for a recognition of osteopathy. Randomized Clinical trail's (RCT’s) offer a "gold standard" for answering these questions, but are no longer "THE golden standard" (De Cock, 2015; Hordijk, 2011). For example 'Clinical Effectiveness Research (CER)’ and the 'Stepped Wedge Design' are two alternative forms of research which are currently rising.

Magid David, principal investigator of the Colorado Permanente Medical Group, took advantage of a CER and applied this to thousands of patient records. He selected patients who tooked both diuretic drugs as well as an Angiotensine Converterend Enzym (ACE)-inhibitor or a beta blocker, yet still had a high blood pressure. He compared gender and age combined with blood pressure along with another row of characteristics in addition to a diuretic and ACE-inhibitors. He linked this to a patient with exactly the same characteristics, diuretics and beta blocker. Then he compared the drugs that worked best. In this way, he prevented a lot of bias. The research took less time to complete compared with a RCT and was much cheaper (Hordijk, 2011).

Research as 'Standardised Data Collection within osteopathic practice’ from the University of Brighton (SDC-Project) in the UK (Fawkes et al. 2014) and 'The

17

Osteopathic Survey of Health Careâ&#x20AC;&#x2122; (OSTEOSURV) in the USA (Licciardone et al.

2001; Licciardone et al. 2011; Licciardone, 2003) are resources that give an insight into collected data of clinical results of a certain osteopathic care.

This study belongs to the RILHOU-RCT, a line of research where the effect of specific indirect osteopathic techniques is examined on the autonomic nervous system (ANS).

The RILHOU-RCT is executed by seven researchers, which handle the same methodology. The line of research 'RILHOU' is not attached to a University or Institute (FICO) and was constituted on behalf of the initiative of the researchers.

The aim of this study is to introduce a scientific study in Belgium within osteopathy in accordance with the declaration of Helsinki, the International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use, ICH-GCP guidelines and national legislation. ICH-GCP guidelines are primarily designed for clinical research within the context of pharmaceutical research, but can be used as a concept within osteopathy (Kaiser, 2013). These guidelines serve for producing scientific research under strict conditions (Robert, 2003). In this study, the emphasis lays on the correct handling of the methodology (submitted to an FMEA) and takes into account possible side effects (adverse events).

This study investigates the immediate and short-term (treatment) effect of a set of indirect osteopathic cranial techniques on the ANS in healthy Dutch adults under optimal conditions. The purpose of this study is specifically aimed to the question "Have indirect osteopathic cranial techniques an impact on the examined parameters?" and not to an explanation model "How the applied techniques works?". The study investigates the hypothesis that the applied techniques have no immediate or shortterm (treatment) effect on the following parameters: HRV, SCL, RSA, PEP and blood pressure.

HRV is taken as primary effect size while SCL, RSA, PEP and blood pressure act as secondary effect sizes. Blood pressure serves as only clinical effect size, while the other parameters belong to intermediate effect sizes.

18

This dissertation consists of seven parts: -

Part I:

A descriptive study of the Belgian legislation on experiments on The human person.

-

Part II:

Heart Rate Variability

-

Part III:

The VU-AMS device

-

Part IV:

A descriptive comparison of the HRV parameters between Software program VU-DAMS (v3.9) and Kubios (v 2.2).

-

Part V:

Methodology and measurements.

-

Part VI:

Results

-

Part VII:

Discussion

19

4 Part I: legislation on experiments on the human person 4.1 Historical review 4.1.1 The Nuremberg Code (1947) In 1947 the Nuremberg Code was created as a result of irresponsible studies mainly conducted by German researchers during World War II. In the literature, also referred to the Nuerenberg Code (ter Heerdt, 2000). The Nuremberg Code was one of the first texts that referred to experiments on humans and was created as ethical counterpart on a court ruling by an â&#x20AC;&#x2DC;American Military Tribunal' (Marrus, 1999). This code is generally regarded as the first document with rules for human experiments on the basis of an informed consent. Some critics claim that the first directives related to experiments in humans would be made around 1900 by the Prussian government.

These guidelines were based on principles of autonomy and represented an earlier model of informed consent. Subjects were informed about possible negative consequences of an intervention, and an understandable informed consent became a mandatory standard. In 1931 the Reich Government obligated a patientsâ&#x20AC;&#x2122; informed consent when performing an investigation. Only in case of a medical emergency and in the interest of the patient it was not operative (Vollmann and Winau, 1996). However, this rule was not follow in World War II. American judges constructed the Nuremberg code which described 10 ethical basic principles for performing clinical trials on humans (Out et al. 2014).

The 10 basic ethical principles of the Nuremberg code: 1. Required is the voluntary, well-informed, understanding consent of the human subject in a full legal capacity.

2. The experiment should aim at positive results for society that cannot be procured in some other way.

3. It should be based on previous knowledge (like, an expectation derived from animal experiments) that justifies the experiment.

4. The experiment should be set up in a way that avoids unnecessary physical and mental suffering and injuries.

20

5. It should not be conducted when there is any reason to believe that it implies a risk of death or disabling injury.

6. The risks of the experiment should be in proportion to (that is, not exceed) the expected humanitarian benefits.

7. Preparations and facilities must be provided that adequately protect the subjects against the experiment’s risks.

8. The staff who conduct or take part in the experiment must be fully trained and scientifically qualified.

9. The human subjects must be free to immediately quit the experiment at any point when they feel physically or mentally unable to go on.

10. Likewise, the medical staff must stop the experiment at any point when they observe that continuation would be dangerous.

4.1.2 Kefauver-Harris Amendment (1962) Following the Thalidomide scandal in 1962 the Kefauver-Harris amendment was introduced. Before the introduction of the amendment the pharmaceutical companies just had to prove that their medicine was safe (Greene and Podolsky, 2012). In 1957, the medication ‘Thalidomide (Softenon)’ was available on the market, even though the Food and Drugs Administration (FDA) didn’t gave approval of grant. This sleeping medication was also used for treating morning sickness in pregnant women. The intake of ‘Softenon’ during pregnancy led to more than 10.000 deformed babies. Testimonies showed that the subjects were not informed that Thalidomide was not approved by the FDA and that the granting of informed consent was lacking (Out et al. 2014). The Kefauver-Harris amendment strengthened the US FDA for the control of experiments on humans and changed the way new drugs were approved and regulated (Webb, 1963). It introduced various directives such as a “proof of effectiveness" requirement, which previously was not present, and an informed consent. Also to explain side effects and effectiveness of the treatments had to be reported to the FDA (Greene and Podolsky, 2012).

21

4.1.3 The Helsinki Declaration (1964) In 1964, the Declaration of Helsinki was conceived by the World Medical Association (WMA). The Helsinki Declaration arose in response to the Nuremberg Code that was regarded as ‘too limited’ and ‘too one-sided’ by the doctors associations (ter Heerdt, 2000). The Helsinki Declaration already described the following: "medical progress is based on research that ultimately must include studies involving human subjects" (WMA, 2001). The purpose of this statement is to direct doctors and other stakeholders involved in clinical research in the field of ethical principles (CCMO, 2009). Some issues in the Helsinki Declaration are a repetition of the Nuremberg Code, for example: the importance of the test subject is paramount and weighs heavier than the importance of the research, voluntary participation of the test subject, .... A number of aspects were added such as: the distinction between therapeutic and non-therapeutic research, the recommendation of a written informed consent, review of research by independent committees, respect for the individual, the right to informed consent ...

(Out et al. 2014; WMA, 2001; Millum et al. 2013; Emanuel, 2013).

The Declaration of Helsinki was repeatedly changed. In 2008 the statement clarifies the admission of placebo use under certain limited conditions. 

The concept of "placebo" is mainly reserved for research with medication. In addition to the credibility, a negligible treatment effect is suggested as a requirement for a Sham intervention for manual therapy. Sham interventions with negligible treatment effects may be unfit for manual therapy studies. The literature suggests placebo as follows: “The focus has shifted from the ‘inert’ content of the placebo agent (eg, starch capsules) to the concept of a simulation of an active therapy within a psychosocial context.” Accordingly, a conceivable Sham intervention for manual therapy is likely to produce some positive treatment effect (Bialosky et al. 2009).

On 19 October 2013 the last change was applied. The main amendments concern the protection of vulnerable groups and the compensation and treatment of subjects in the case of damage caused by the research. Extra attention is demanded to minimize and monitor the risks to subjects during the examination. Also the obligations after the end of the investigation are expanded. There is, among other things, the obligation of reporting the study results to the Committee Medical Ethics (CME) and the provision of proven interventions beneficial to the subjects after the investigation (WMA, 2013). The Helsinki Declaration is an important document. The ‘Universal Declaration of human

22

rights (UDHR)' and ‘EVRM’ (De Pooter. 2002) as Royal Decrees (K. B. ‘s) refer to it.

The Helsinki Declaration is not a legally enforceable instrument under national or international law, and its influence as a policy instrument is controversial. This statement is only a code of ethics (Pierik, 2006). Ter Heerdt J. describes in her book ‘Het experiment beproefd: een juridische analyse van medische experimenten op de mensen.…', that some authors consider that Directive 19/507/EG from the Royal Decree of 22 september 1992, requires explicit compliance (ter Heerdt, 2000).

4.1.4 ICH-GCP The United States and some EU countries designed more detailed guidelines, which led in 1990 in the EU to the European Good Clinical Practice (EU-GCP). GCP guidelines are international, ethical and scientific quality standards in the design, conduct, performance, monitoring and reporting of clinical trials. These directives guarantee the rights, integrity, and confidentiality of the test subject, as well clarifies the responsibilities and mutial expectations of any other stakeholders (principal investigator, sponsor, …) included in the clinical trial. This resulted in 1996 in the ICHGCP. The conversion into a European legislative framework followed in 2001, known as 'Clinical trials guidelines Eudralex, volume 10’ (Verrijken, 2008). EurdraLex stands for the set of rules and regulations governing medicinal products in the European Union. The most important aspects of the ICH-GCP guidelines can be described as follows (Directive 2001/20/EG): 

The conduct of a clinical trial must be in accordance with the ethical principles as described in the “Declaration of Helsinki".



Prior to the start of a clinical trial the expected risks and inconveniences must be weighted against the anticipated benefit for the individual subjects and society. A clinical trial may only be performed and continued as the expected benefits justify the risks.



The rights, safety and well-being of the trial subjects are the most important considerations and are primarily above the interests of science and society.



The available preclinical and clinical information on an investigational product should be sufficient to the research proposal.

23



Clinical research must be scientifically justified, and described in a clear, detailed protocol.



The clinical investigation must be performed in accordance with the protocol on which in advance a positive opinion and approval has been obtained from a CME.



The medical care and medical decisions for the benefit of the subjects are always under the responsibility of a qualified physician or, if applicable, a qualified dentist.



Any individual involved in the conduct of a clinical study must be qualified by education, training and experience to his or her respective task/tasks.



Prior to his participation in a clinical trial the "informed consent" from each volunteer or patient should be obtained, which is entirely voluntary. The patient may discontinue participation at any time without penalty or loss of benefits to which the patient is otherwise entitled, without that this decision has any effect on the quality of any (further) medical care.



All information concerning the clinical trial must be recorded, handled and stored in such a way that it is available for accurate reporting, interpretation and verification.



The confidentiality of the documents to which subjects can be identified should be protected, where the rules for privacy must be respected, in accordance with the relevant legal requirements.



The resources should be manufactured, handled and stored in accordance with the relevant Good Manufacturing Practice (GMP).



There should be systems and procedures that ensure the quality of every aspect of the research.

These guidelines ensure that different national procedures were harmonized and that the various member states were obliged to provide a basic protection for each participant in a clinical trial. The procedure for clinical trials and the required basic

24

protection for the participants was standardized. A novelty in Directive 2001/20/E is article 3. ยง2f: "A clinical trial may be undertaken only if, in particular, is provided in the insurance or cover the liability and responsibility of the investigator or sponsor". Taking into account are the possible damages and the resulting liability that the organization/researcher of the medical experiment bears.

Through the Directive 2005/28/EG, article 3. ยง1, the European Member States obtained the obligation to implement the previous directive by 29 January 2006 in their national legislation. Belgium implemented Directive 2005/28/EG by the K. B. of 30 June 2004 to provision implementing measures scheme on the law of 7 May 2004 concerning experiments on the human person (B. S. 18 May 2004).

4.2 The Belgian legislative framework concerning experiments on the human person 4.2.1 Law of 7 May 2004 concerning experiments on the human person (B. S. 18 May 2004) This law consists of 25 chapters and describes 36 articles. Up to this present date this law was several times modified by means of program laws, laws and K. B.'s which were published in the B. S. The last amendment in the Belgian Official Journal with regard to the law of 7 May 2004 was on 26 December 2015 (Law programme No. 2015205966, 80610). The last text revision is dated at 27 December 2016.

The approved design of the law of 7 May 2004 was submitted by the Council of Ministers on 19 December 2003 to the Council of State for advice. The Council of State presented the submission over to the National Council. The National Council disagreed that the extension of Directive 2001/20/EC of the European Parliament and of the Council of 4 April 2000 was positive [concerning the approximation of the laws, regulations and administrative provisions of the member states relating to the implementation of GCP in the conduct of clinical trials on medicinal products for human use to all experiments] (Orde der artsen, 2004).

From 1 May 2004, the European Directive 2001/20/EG was binding. Belgium published on 18 May 2004 in the Belgian Official Journal (B. S.) the law of 7 May 2004 concerning experiments on the human person. Article 36. ยง1 stated that the law would

25

be operative on 1 May 2004. In this way the law was retroactively inserted and confirmed to the European Directive 2001/20/EG (Dresse, 2005). The Belgian legislature chose a more extensive protection than the one which was included in Directive 2001/20/EG. Beside clinical trials also medical tests outside the drug research field were subject to the new law of experiments (Pinxten et al. 2008). In article 2. 11° "experiment" is stated as: “every on a human person conducted trial, study or research, with an interest to develop the biological or medical knowledge” (B. S. 18 May 2004).

The court of arbitration annulled on 16 November 2005 in article 2. 11° the passage 'study or research', but it is still quoted in the law programme (De Cock, 2016).

The federal legislature in turn modified through a law programme on 27 December 2005 the scope of article 2. 11° in the following sentence: "Experiment: every on a human person conducted trial, study or research, with an interest to develop the knowledge specific to the exercise of the health care professions referred to in the Royal Decree n° 78 of 10 November 1967 on the exercise of the health care professions". Referring to the K. B. n° 78 indicates that the law on the experiment is a federal matter. This implies that the K. B. n° 78 of 10 November 1967 was possibly manipulated after signature by the King (De Cock, 2016).

4.3 Application of the law of 7 May 2004 4.3.1 Chapter I: General provision Article 1 defines that this law a affair settles as referred in K. B. n° 78 of 10 November 1967. The definitions in the law of 7 May 2004 and in the K. B. n° 78 of 10 November 1967 on the exercise of the health care professions are on the decision of the law of 7 May 2004 applicable (article 1. §1).

For non-conventional practices concerning to medicine, pharmacy, physical therapy, nursing and paramedical professions is the law of 29 April 1999, (the so called Law Colla) involved. 

The law Colla considered osteopathy as 'non-conventional practice' (Article 2. 2°).

This law provides measures for the legitimate practicing of non-conventional practices. Article 8. §1 refers that nobody can practice one of the registered nonconventional practices before one is registered by a recognized professional association (Law of 29 April 1999).

26

In article 1 of the K. B. 10 February 2003, four Osteopathic professional associations were recognized: Union of Osteopaths (U. V. O), Belgian Union of Osteopaths (BUO), Belgian society of Osteopathy, and the Register of Osteopaths of Belgium (R. O. B). The K. B. of 10 November 2005 mentions the Union of graduates in the physiotherapy and Osteopathy (U. K. O) while the Belgian Association of classic osteopaths (BAKO) were recognized in the Royal Decree from 6 April 2010. By means of derogation from the first paragraph, the professional person who has submitted an application for registration within a period of 6 months after publication in the Belgian Official Journal (as referred in article 3. §3 taken measures), can continue practicing the non-conventional activities (Law of 29 April 1999). The Minister of health (De Block Maggie, legislature Federal Parliament 2014 - 2019) must make a declaration within 12 months on the request of registration (Article 8. §1, law of 29 April 1999).

Article 8. §2 refers that registration is given out by the Minister of Health on the advice of the relevant Chamber. Registration is granted if the concerned person corresponds to all of the terms and conditions for the exercise of non-conventional practices in accordance with the provisions of K. B. of 26 March 2014. On exception of doctors, article 10 refers that nobody is allowed to perform any surgical

procedures,

nor

give

any

injection

of

medicines,

nor

sedate

pharmacological patients (K. B. 26 March 2014). The Consumer Council gave on October 25 2007 a negative assessment on the application for protection of the title 'osteopath', 'osteopath D. O.' (Diploma in Osteopathy) and 'osteopath DO’ (Doctor of Osteopathy) (RvV 392, 2007). ‘The difference between D. O. and DO was not mentioned in the application for the protection of the title of ... '. According to article 2 of the K. B. n° 78 nobody is allowed to exercise medicine if one doesn’t possess the legal degree of doctor in medicine, surgery or obstetrics. The degree should be achieved in accordance with the legislation on the awarding of academic degrees and the program of the University exams. If the conditions are not fulfilled in accordance with article 7. §1 or §2, the law considers this as an illegal practice of medicine (K. B. n° 78 of 10 November 1967). In the motivation request about protection of the professional title ‘osteopath’, also the practice of the profession osteopathy is explicitly requested. The Consumer Council emphasized that acting without a prescription on the domain defined as 'medical' is an infringement on the K. B. n° 78 on the practice of the health care professions (RvV 392, 2007).

27

4.3.2 Chapter II: Definitions and application This chapter describes article 2 and 3 from the law of 7 May 2004. Article 2. covers the application of the law and how it should be understood. Here is the investigation file being described as clinical and non-clinical data on the investigational drug or drugs that are relevant to the study of the medicine or the investigational medical products (Article 2. 3°). 

Each subject has an investigation file in the RILHOU-RCT (De Cock, 2014). The file contains a cover page where the file number (file code) of the test subject is noted. By means of this file code personal data can be placed in connection with the identification of the test subject (Article 1. 3°; 7. K. B. 13 February 2001). The code of the researcher, the number of the subject, the date and time of the research as well as the randomization range of the test subject are noted. On the cover page of the investigation file, yes/no is marked for the subject that can be included or excluded in the research. The investigation file contains: administrative data, a randomized questionnaire and a registration form where all parameters are noted for the experiment and follow up. External variables such as room temperature, relative humidity percentage and environmental noise are also included. The investigation file contains three separate pages for the registration of adverse events, an ICF (Addendum 1), a signed letter of agreement and a consent form document (Addendum 2). In the investigation file is also an extra page included in case of participation withdrawal from the trial. At the end of the file are the results obtained, noted with file code (Article 9, K. B. 13 February 2001). At every page the file code is noted (De Cock, 2014).

To comply with the legal obligations to perform an experiment on a human person a approval by a fully recognized CME is required (Article 5. 6°, B. S. 18 May 2004). A distinction is made between 2 types of ethical committees. The K. B. of 16 May 2014 to determine the implementing measures of the law of 7 May 2004 on experiments on the human person (in relation to the ethics committee) defines in article 1. §2. 3° that the coordinating ethics committee shall give its opinion in the case of a monocentric trial, or just advice in the case of a multicenter trial. The local ethics committee is linked to the location or the structure where the experiment is carried out and shall not-only opinion on points 4°, 6° and 7° in article 11. §4 (law of 16 May 2014). 

On the advice of Dr. Stevens, President of CME to Rocour hospital, (CME with partial recognition) the request for advice to the RILHOU-RCT to assess on its

28

ethical aspects and privacy (subordinate clinical relevance) was submitted to the CME Hospital East-Limburg (ZOL) (De Cock, 2014). This is the coordinating CME for the RILHOU-RCT, because the experiment was performed monocentric (FICO, Santvoortbeeklaan 23, 2100 Deurne, Belgium) (Article 2. 13°, B. S. 18 May 2004).

This study belongs to a non-commercial experiment (Article 2. 14°, B. S. 18 May 2004) and was executed independent of a University/ institute (FICO) or hospital (De Cock, 2015).

The CME is an independent authority and consists of experts from the healthcare and members from outside the medical world (Article 2. 4°, B. S. 18 May 2004; Article 4, B. S. 16 May 2014). The CME shall ensure the protection of the rights, safety and wellbeing of the subjects who participate in a clinical trial. The CME judges on the protocol of the trial (Article 2. 22°), the suitability of the researchers, the facilities and the methods and documents used to inform the test subject (informed consent form) and their informed consent (consent form) (Article 2. 4°). The solely assessment of the coordinating CME must expressly and adequately motivated. All the legal and factual considerations should be included in the opinion. The opinion makes a final judgment that can be positive, negative or positive under certain conditions (Article 2, B. S. 16 May 2014). In the solely advice of the CME are different elements expressed forinstance: a precise description of the locations where the solely advice refers to (Article 2. 1°, B. S. 16 May 2014). In article 2. 5° the motivation is explained why the experiment for which the application is submitted falls under the scope of the law (B. S. 16 May 2014). 

In the questions designed by the CME ZOL the competence of the researchers and employees was questioned.



To improve the quality of the researchers in RILHOU-RCT the following enforcement measures were guaranteed (De Cock, 2014): 

Standardization of the modus operandi was described in the ‘Common documents’ (De Cock, 2014) and in the 'RILHOU-RCT DRAAIBOEK + FMEA (werkdocument)'.



Techniques were regularly practiced under supervision of the associate supervisor, according to a clearly described treatment protocol (De Cock, 2014).

29



The treatment protocol was regularly practiced in group.



Techniques were applied by the researchers in their own practice.



In case of problems with the HRV software (VU-DAMS) advice could be obtained from experts of The Free University of Amsterdam.



Continuous training with regard to the establishment of a good research in accordance with the ICH-GCP guidelines, statistics, etc., was led by De Cock JJ (scientific coordinator for the RILHOU-RCT).



Collect the correct data and quality control procedures were granted.



To perform an experiment, there are four essential characteristics: experimental manipulation, standardized procedures, control and surveillance over all variables except the independent variables and a random allocation to the conditions or order (Howitt and Cramer, 2007). The software-generated randomization that was applied in the RILHOU-RCT does not cover the concept of "random". The RIHOU-RCT uses a strict methodology (see infra) (De Cock, 2014).



All participants were screened for participation in the study by using a randomized questionnaire (inclusion and exclusion criteria).



External variables such as: ambient temperature, relative humidity percentage (Ren et al. 2011; Gold et al. 2000), and environmental noise (Sim et al. 2015; Kraus et al. 2013) were observed and noted during the experiment and followup.



The population sample was determined by inclusion and exclusion criteria.

Through software-generated randomization with blind allocation the sample was divided into 2 groups (experimental group and Sham). The follow-up took place 6-10 days after the experiment (Addendum 3). There was aimed to optimize the equipoise principle (Turner, 2013; Mann, 2011; Stehouwer et al.

2010; De Cock, 2014).



The subjects, researcher and statistician were blinded (Infra: 8.3.7.4 Blinding).

30

Article 2 describes some definitions such as: side effects (Article 2. 5°), unexpected side effects (Article 2. 6°), clinical trial (Article 2. 7°), adverse event (Article 2. 9°), serious adverse event or serious adverse reaction (Article 2. 10°). Side effects, adverse reactions, serious adverse events or serious side effects are only related to experiments/trials with medication. The law of 7 May 2004 refers to an 'adverse event' when a harmful phenomenon occurs in a patient or subject in a treatment group during the trial. This article does not specifically describe that an 'adverse event' only relates to drugs. In preventing and the occurrence of an 'adverse event' there doesn’t necessarily have to be a causal link with the experiment (Article 2. 9°). In the medical literature, there are several definitions about adverse reactions and events of medicinal interventions (Goldfarb, 2012). Side effects and adverse events are drawn from pharmacological vigilance. The WHO defines adverse events as: "Any untoward medical occurrence that may present during treatment with a pharmaceutical product but which does not necessarily have a causal relationship with this treatment". In the literature there is no unanimity in the common terminology for adverse events (Vogel et al. 2012).

In 2009, researchers have sought to create a framework for the exploration of the incidence and prevalence of adverse events in manual therapy (Carnes et al. 2010b). A hierarchical system was used to define and categorize possible adverse events. The authors concluded that classifying adverse events was difficult without the context of the details. Classification of adverse events can be improved on the basis of taxonomy and description. They came to a pragmatic definition of adverse events in particular: 'severe', 'moderately' adverse events and 'mild' and 'non-negative' adverse events (Carnes et al. 2010b; Carlesso et al. 2015). 

Side effects were observed and noted in the RIHOU-RCT as 'adverse events'. This for different time periods and during the follow-up. The measuring researcher observed and noted the adverse events for the pre-measurement, postmeasurement and follow-up. During the follow-up the measuring researcher made an inventory of adverse events that occurred in the past days after the experiment.

The researcher who performed the experimental techniques/Sham observed only adverse events when performing the intervention. This researcher had no access to the investigation file to the end of the cohort. (De Cock, 2014).

Article 2 paid attention to the ordering party, in clinical literature often referred to as ‘sponsor’. This can be a person, company, institution or organization which takes

31

responsibility for the initiation, management and/or financing of the experiment (Article 2. 21°). The ordering party is liable for the faultless damage that the subject (or in the case of death, his owner) has accumulated, which have a direct or indirect link with the experiment. Any contractual provision to limit this liability is deemed null and void (Article 29. §1). 

During the advice-request at CME ZOL it was mentioned that there was no ordering party for the RILHOU-RCT. The study is on own initiative of all researchers within the framework of achieving their diploma of Master in the osteopathy.



Each researcher has been individually informed in writing his insurance with the participation as a researcher and execution of the study (De Cock, 2014).



The Medical Director of the ZOL (Dr. Van der Velpen) has submitted a request to insurance company Ethias concerning an insurance certificate for the RILHOURCT, performed by externals. Addenda were included to the request: RILHOURCT submission letter to CME ZOL; Request for advice RILHOU-RCT to CME ZOL; Document technical sheet East-Limburg hospital.



Germeys K (service responsible production civil liability) of insurance company Ethias (Prins Bisschopssingel 73, 3500 Hasselt) corresponded on 2 September 2014 by email to Wens C (contact person RILHOU-RCT) and Dr. Van der Velpen (in copy) that Ethias could not provide the compulsory insurance for the study because of the lack of clarity of the ordering party.



A second request to insurance company ‘Ethias’ was submitted by mail on the same day (2 September 2014) for regarding "VZW Health" as ordering party for the RILHOU-RCT. On 10 September 2014, a policy proposal (nr. 45.326.303) was proposed by Ethias Insurance of civil liability for experiments on a human person, after personal consultation with the contact person of the research (Wens C).



A second insurance company 'Assura Portima' (Lange Nieuwstraat 21 Bus 237, 2000 Antwerp) was informed by email on 2 September by Wens C related to the compulsory insurance. This request served both for ordering party 'VZW Health' as for Wens C (individual trial). On request of 'Assura Portima' (by phone) an e-mail was sent on 6 September 2014 with the addendum 'advice request insurance policies of all the participating researchers'.

32



On 9 September 2014, 'Assura Portima' gave a telephonic negative advice for taking out a compulsory insurance (for ordering party “VZW Health” and individually for Wens C). Assura Portima described that insurance company ‘AMMA’ and ‘Belfius Insurance’ did not wish to grant the compulsory insurance.



The CME ZOL mentioned in the preliminary negative advice of 10 October 2014 that for the study there must be a responsible legal ordering party and from a legal point of view it was obligatory to close the compulsory insurance (De Cock, 2014).



To comply with article 29. §2 and article 5. 9° “VZW Health” was appointed by the RILHOU-team as ordering party for the study (De Cock, 2014). CME ZOL accepted “VZW Health” as official ordering party for the RILHOU-RCT. This implies that the rights and obligations for the RILHOU-RCT belong to ‘VZW Health’.



Ethias insurance civil faultless liability for “VZW Health” concerning experiments on a human person for the RIHOU-RCT. The original validity period of the policy (15 December 2014 – 15 December 2015) was extended until 31 December 2016.

Article 3 describes the application to experiments on a human person, but is not applicable on investigations without intervention (Article 3. §1) and retrospective studies based on data from the past. The CME may formulate objections around: 1) the expected benefits on therapeutic and public health, taking into account the risks and inconveniences for the participant (Article 3. 1°, B. S. 16 May 2014); 2) the relevance of the clinical trial (Article 3. 2°); 3) the reliability and size of the clinical trial, which takes account of the statistical approach, trial design as well as with the methodology (Article 3. 3°); 4) the security measures including monitoring and security reporting (Article 3.

4°); 5) the completeness and adequacy of the investigation file (Article 3. 5°). 

The CME ZOL noted in at 10 October 2014 with questions and comments that this was the first time to assess a study within osteopathy on the ethical and legal aspects for the CME. The reply on the temporary negative advice (with three fundamental objections and 41 questions and/or comments) has led to a final positive advice of the CME, on date of 9 December 2014 (De Cock, 2014; De Cock, 2015).

33

4.3.3 Chapter III: General provision on the protection of participants Experiments and trials must be designed, applied and disclosed in accordance with quality requirements, this for ethical and scientific domains. These requirements are internationally recognized and must be respected during the planning, application, registration and publication of the experiments (Article 4). 

In Belgium, there is no legal obligation for a study to register at an official website.

The CME ZOL recommends to register the study at an official website. This study was announced by the coordinator dissertations FICO, Dr. Quaghebeur, PhD, MSc, D. O.



Before the start of the trial, the study protocol together with the request for advice CME ZOL, provisional advice CME ZOL, reply to questions and comments by the CME ZOL, documents Ethias insurance and additional correspondence were legally deposited in the form of a 'Common documents' in the Royal Library in Brussels (De Cock, 2014). In this way all deposited data are publicly accessible so anyone can review the modus procedendi (operandi). This makes external control possible (abinitium) (De Cock, 2014).



The RIHOU-RCT was announced in a Dutch specialist journal 'De Osteopaat' on the basis of an article that described a review of (osteopathic) dissertations about the effect of (cranial) manual techniques on the heart rate variability (HRV) and compliance with the ethical requirements (De Cock, 2015).

A trial may only be commenced or continued under certain conditions (Article 5). The experiment must be scientifically justified and based on the latest state of scientific knowledge and on a sufficient pre-clinical experiment (Article 5. 1°). 

Both in Europe and in the rest of the world cardiovascular diseases still cause a great morbidity and mortality (Nichols et al. 2012; Nichols et al. 2013; Santulli G, 2013). HRV analysis has become an important diagnostic tool in cardiology (Billman et al. 2015). From scientific literature emerges the relevance to examine the significance of HRV. In databases such as PubMed or Medline there are thousands of references on HRV (Sassi et al. 2015).

34



Registration of HRV and electrodermal activity is a non-invasive way to evaluate the sympathovagal balance (Sztajzel, 2004; Kleiger et al 2005; von Borell et al.

2007; Benedek and Kaernbach, 2010; Bach et al. 2010; Sawyer et al. 2015; Bonnemeier et al. 2003). An imbalance in the autonomic nervous system characterized by a hyperactive sympathetic nervous system and a hypoactive parasympathetic nervous system is associated with various pathologic conditions (Thayer et al. 2010; Kiecolt-Glaser et al. 2002; Boer-Martins et al. 2011; Berntson and Cacioppo, 2004; Friedman and Thayer, 1998).



In 2010, The World Health Organization outlined a document ‘Benchmarks for training osteopathy' where scientific research within osteopathy is recommended (World Health Organization, 2010; Moore et al. 2014).



In 2015, De Cock studied 18 dissertations of osteopathy (national and international) related to the ethical and legal requirements (ICF, consent form, request for advice and approval of 'CME'. Only 22.2% (4 dissertations) was found satisfactory concerning to the ethical and legal guidelines (De Cock, 2015).



HRV parameters are obtained through an electrocardiogram (ECG) in accordance with the guidelines of the TASK FORCE OF THE EUROPEAN SOCIETY OF CARDIOLOGY AND THE NORTH AMERICAN SOCIETY OF PACING AN ELECTROPHYSIOLOGY (Task Force, 1996a,b; Sassi et al. 2015).



The VU-AMS device is a valid non-invasive ambulatory monitoring system developed by the Free University of Amsterdam (Parry MJ and McFetridge-Durdle door, 2006; de Geus et al. 1995; de Geus and van Doornen, 1996; Willemsen et al. 1996; Riese et al. 2003; van Doornen et al. 2009). The device registers ECG/ICG (impedance cardiogram) and electrodermal activity.

The objective of the experiment is to extend the knowledge of the human being, thus by extending the means in which his condition could improve (Article 5. 2°). 

The design of the RILHOU-RCT is to formulate statements about the efficiency and effectiveness of the applied indirect osteopathic techniques on HRV and electrodermal activity (SCL). An intermediate purpose is to obtain or to demonstrate changes in the intermediate effect sizes. Effect-sizes learn a little more about the physiological/ physio-pathological changes in the ANS. Blood

35

pressure is the only clinical effect size (final objective). Here are specifically considered whether the applied techniques have an influence on blood pressure in healthy adults (between 20 and 50 years old) and on the other hand in adults with obesity (De Cock, 2014). 

This study belongs to an efficiency research (explorative research) and examines whether of indirect osteopathic cranial techniques (under optimal conditions) have an immediate or short-term (treatment) effect on the chosen parameters. ‘Does it work or doesn't it work ' (De Cock, 2014).



An effectiveness research (pragmatic study) examines whether the applied techniques (not under optimal conditions) may have a positive effect on the chosen effect sizes. This covers the practical generalizability, i. E. the average practice situation. The total RILHOU-RCT is partly of clinical epidemiological nature and could possibly belong to a causal experimental, longitudinal cohort study (Stehouwer et al. 2010) by means of establishing the standard values of HRV (possibly SCL and PEP) for the studied groups (De Cock, 2014).

Article 5. 3° indicates whether there is no alternative method that exists whose effectiveness is similar and allows to achieve the same results. Foreseeable risks and disadvantages (mainly by physical, psychological, social and economic nature) were weighed against the individual benefit to the participant in question. Also what concerns their right on the respect of the physical and psychological integrity; the right to respect of their personal situation in life and protection of personal data (Article 5. 4°). The evaluation leads to the conclusion that the expected benefits on therapeutic and public health surpass their risks. The experiment may only be continued to such an extent that there is constant surveillance of compliance with all the specified requirements.

Interests of the participant obtain priority over the interests of science and the community (Article 5. 5°).  Risks and benefits were established. The investigation focuses exclusively on healthy adults, one sub-trial focuses on obesity adults. Contraindications with regard to the implementation of the applied techniques were included in the exclusion criteria. The techniques that are applied in this study belong to indirect osteopathic techniques, hands-on techniques were used in the Sham group.

Manipulations (in accordance with European definition) are not applied in the RILHOU-RCT. So far, no risks and/or side effects are known or described in the literature for the applied techniques in this study, but these are not excluded. In

36

case side effects occur, they will be noted in the investigation file and be treated most accurate. 

In Chapter 1, 2 and 3 of the document 'RILHOU-RCT DRAAIMAP + FMEA (werkdocument)' possible risks and safety measures were taken into account. For this purpose a proactive risk and safety analysis was used based on a FMEA and subsequently controlled by an external safety and prevention advisor. FMEA is an inductive method and systematic technique for prospective failure analyses to study problems that might arise from malfunctions of a system. Different components and subsystems are critically reviewed to identify potential failure modes, their causes and effects. It identifies components vulnerable for human factors and influences. In the RILHOU-RCT a FMEA is combined with a semistructured observational registration of benefits and harms coupled with a simple questionnaire. The FMEA was conceived by the contact person of the RILHOURCT (Wens C) in collaboration with an external safety advisor (Janssens P). A retroactive analysis wasn’t performed for this trial (De Cock, 2016).



The FMEA was tested in a preliminary study. The improved final version was used in the investigation (De Cock, 2016).



Before the start of the study the subjects had the opportunity to inform their family doctor and family about regarding participation in the study (subjects received foregoing the study an email with an attachment specifically aimed to the family doctor).



There are no risks involved in the registration of ECG/ICG and electrodermal activity. Positively, it could be an advantage that by accident an abnormal ECG could be found. The digital ECG’s were given to the subjects after closing this study. At the request of the subject, the ECG can be submitted to the family doctor.

On allergy to isopropyl alcohol, Hibidil was provided as an alternative.



During the experiment the subject is constantly under supervision. A nurse permanence was as good as possible present. The family doctor or the permanence doctor could be contacted if problems arose.

For the legislation on data protection can be referred to the law of 11 December 1998 ‘Act on the protection of privacy in relation to the processing of personal data’, which is

37

in conformity with the European Directive 95/46/EG of 24 October 1995. This law is an adaptation of the original law of 8 December 1992 on the protection of privacy with regard to the processing of personal data (K. B. 13 February 2001; K. B. 17 December 2003).

The European Union provides a legislation on data protection, which consists of some treaties (Treaty on European Union; Treaty on the functioning of the European Union) and describes the data protection as a primary EU law. The regulations, directives and decisions of the EU are often referred to as secondary EU law.

The most important legal instrument of the EU in the field of data protection is Directive 95/46/EG of 25 October 1995. It concerns the protection of individuals with regard to the processing of personal data and the free traffic of the data. Directive 2002/58/EG on privacy and electronic communications was declared invalid on April 8, 2014. With the commencement of the Treaty of Lisbon (1 December 2009) the Charter of fundamental rights of the European Union (which was initially only a political document) was legally binding and thus also a primary EU law (EU, 2012). Article 16 of the Treaty on the functioning of the European Union (VwEU) contains a provision that grants general power to the EU legislation on matters relating to data protection. Claim (EG) no 223/2009 on European statistics (EUR-Lex 52012PC0167) contains essential requirements for data protection in official statistics, and can therefore be regarded as relevant for regulations on official statistics at national level. 

Each subject in the RILHOU-RCT has an investigation file with a file code to preserve the integrity of the person (Article 1. 3°; 7. K. B. 13 February 2001). The subject has the possibility (provided that thoroughly motivated and in dialogue with the scientific coordinator and the researcher) to access to the investigation file.



All the files and data from the RILHOU-RCT will be preserved by ‘VZW Health’ for a period of 20 years after the completion of the investigation, recommended by CME ZOL. This period is longer than the proposed term of at least five years, as meant in article 17 of Directive 2005/28/EG.

The protocol has received an ultimate favorable and positive advice of an CME with full recognition (Article 5. 6°). 

The RILHOU-RCT has obtained an ultimate favorable advice by the CME ZOL (Eudract/B-nr. B371201422023).

38



Every subject has given his written consent before the start of the experiment and has a contact point where more information can be obtained (Article 5. 7°).



Previous to the start of the study, the 'Informed Consent Form’ (ICF) was sent by email to the subjects. When the subject participated in the study, the ICF went through together with the subject before the experiment started at the location of implementation of the experiment. The subject was explicitly asked if he had any questions related to the study and if everything was clear. If the subject had no questions the 'Letter of agreement and consent form' went through. This document consists of two pages, printed on both sides. The study was only started if the subject had no questions related to the trial and an agreement was signed, priory written 'for read and approved'. Also the investigator signed the ‘Letter of agreement and consent form' (De Cock, 2014).



The ICF mentioned under the item 'for specific questions' the following: 'A list of the cooperating researchers will be given, as soon the treatment starts'. The ‘Letter of agreement and consent form' mentions that: 'I have the opportunity to ask questions to the responsible investigator about his identity, accessibility and every aspects of the study...'.



The ICF was not stapled to the 'Letter of agreement and consent form’ due to ecological and financial considerations. All the documents were sent in advance by e-mail to the subjects, only on demand of the subject a printed version of the ‘ICF’ and 'Letter of agreement and consent form’ was given (De Cock, 2014).



During and after the study the subject has the possibility to appeal to the investigator, the coordinator of the research and family doctor (De Cock, 2014).

The provided care and decisions made in relation to the participants are of the responsibility of a qualified practitioner in accordance with the determination of the K. B. N° 78 of 10 November 1967 concerning the exercise of the health care professions (Article 5. 8°). 

On 6/10/2015, The RILHOU-RCT counts five researchers that not have been in the possibility to register with a legally recognized Osteopathic Association in accordance with article 8. § 1, law of 29 April 1999 (see supra 2.2.1. Chapter I:

39

General provision). Two researchers are registered at a recognized Osteopathic Association in accordance with article 8. §1, law of 29 April 1999.

The insurance and the coverage of the liability of the investigator and sponsor are organized in accordance with the provision of article 29 of the B. S. 18 May 2004 (Article 5. 9°). 

“VZW Health” served as ordering party for the RILHOU-RCT and has closed a legal insurance with company Ethias for the entire duration of the study (De Cock, 2014). The insurance and the coverage of the liability of the investigator and sponsor are organized in accordance with the provision of article 29 of the B. S. 18 May 2004 (Article 5. 9°).



The contribution for this insurance for the entire duration of the study was concluded against payment of a single premium of 265,00 Euro.



The security deposit was limited to 2.500.000,00 Euro per insurance claim with regard to the physical, tangible and intangible damage. This limit applies also a maximum deposit for the entire duration of the experiment.



The deposits of this policy are only applicable on experiments where the Belgian right is being invoked, on Belgian territory. The RILHOU-RCT was performed at FICO, Santvoortbeeklaan 23, 2100 Deurne, Belgium.

In an experiment, the subject may only participate in so far as he/ she has agreed on a free and informed way. With the exception of the persons referred to in articles 7. 8 and 9, with the information referred to in article 6. §2, were notified in advance. This permission must be written (Article 6. §1). Article 6. §2 defines the information referred to in article 6. §1 as follows: 'the nature, the scope, objectives, effects, expected benefits, the risks of the experiment, the circumstances in which it takes place, the identification and the opinion of the competent ethical committee in accordance with the provision of article 11’. The subject or representative as referred in articles 7 and 8, is informed of his right to withdraw at any time. The participating subject can withdraw from the experiment without experiencing any disadvantage (Article 6. §3). This information should be contained clearly, written and in an understandable way in advance to the subject who participates to the experiment or to his representative. The subject or his representative has the option for a conversation with the investigator or a

40

member of the research team for further information (Article 6. §2). 

The ICF consists of 5 pages (Addendum 1). In this document the following items were explained: title of the clinical experiment; general info; specific questions; background of the study; explanation ECG/HRV; importance and type as the purpose of the study/experiment; short summary of the project; nature of the techniques; risks and inconveniences with accompanying measures; implication with regards to the participant; the voluntary nature of participation; benefits and stress of the participant; responsibility for the confidentiality of the data; treatment after the study; costs/financing; insurance; approval CME ZOL (Article 6 §1; §2 and §3; De Cock, 2014). The explanatory statement 'ICF' and the 'Letter of agreement and consent form' were described supra (Article 5. 7°).



The documents 'ICF' and 'Letter of agreement and consent form' were submitted to the CME ZOL (De Cock, 2014). The omission of a Sham approach in the ICF was twice explicitly submitted to the CME, but went unanswered. By the final approval of the ICF one implicitly gave their agreement/ permission, also for not mentioning a Sham approach in the ICF.

4.3.4 Chapter IV: Special provision for the protection of minors taking part in experiments An experiment with minors (underaged subjects) is only allowed under strict conditions, without prejudice to compliance with the conditions as stated in the article 5 and 6.

These conditions are specifically described in 8 points under article 7.

4.3.5 Chapter V: Special provisions for the protection of adults who are unable to give their consent for participation in experiments With the clauses of article 5 and 6 a trial with an adult person that is protected under article 492/1 of the civil code is only allowed under specific conditions (Article 8, B. S. 18 May 2004).

41

4.3.6 Chapter VI: Special provisions for the protection of

persons whose consent cannot be obtained because of urgency When the permission cannot be obtained because of extreme urgency, the experiment may only be conducted under specific conditions. These conditions are described in six points under article 9. 

Article 7, 8 and 9 do not apply to the RIHOU-RCT because of the target group belonged to healthy mentally competent adults (De Cock, 2014).

4.3.7 Chapter VII: Beginning of the experiment An experiment may only start under the condition that the ordering party and investigator have a favorable assessment of a [fully recognized] CME, in accordance with the provision of Chapter VIII of the B. S. 18 May 2004 (concerning experiments on the human person). A departure from the first paragraph is sufficient in order to obtain a favorable advice by a [partially recognized] CME taking into account article 11. §4, first paragraph, 1° to 11° of the Belgian Official Journal of 18 May 2004 (Article 10).

Further, it is provided that it’s about a non-interventional monocentric experiment, as referred to article 2. 8° (trial without intervention). It must be done in the framework that is required for the acquisition of a Bachelor’s degree (Article 10. 1°). The partially recognized CME is linked to the location where the experiment takes place. In case of a trial this only may start on the condition that the Minister of Health has made no knowable objection (Article 10. 2°).



The RILHOU-RCT is set up for to obtain a Degree in Osteopathy and/or a Master of Science in Osteopathy. In Belgium, the title D. O. belongs not to an academic degree and is not protected by the Belgian Government. Hence, the RILHOU-RCT acts independently and is not attached to a line of research of a University/ institute (FICO). It belongs to a monocentric Interventional study. The request for advice was therefore directed to a fully recognized CME (ZOL) (De Cock, 2014).

42

4.3.8 Chapter VIII: Ethics Committee The researcher who wishes to organize a trial in Belgium shall address its request to a fully recognized CME. Article 11 (B. S. 18 May 2014) describes fourteen paragraphs that should be taken into account.

The competent CME phrased its advice and takes into account different aspects (Article 11. §4) such as: 1) The relevance and scope of the experiment (Article 11. §4. 1°).

2) The assessment of the expected benefits and risks as well as the merits of the conclusions, more specifically on therapeutic and health areas (Article 11. §4. 2°).

3)

The protocol (Article 11. §4. 3°) and the ability of the researcher and his staff (Article 11. §4. 4°).

4)

The investigation file (Article 11. §4. 5°).

5)

The quality of the facilities (Article 11. §4. 6°).

6)

The adequacy and completeness of the ICF and the procedure relating to the consent (Article 11. §4. 7°).

7)

The arrangement for compensation and/or compensation when a participant is injured or dies as a result of an experiment (Article 11. §4. 8°).

8)

Surveillance or insurance and other guarantees to comply with the provision of article 29 concerning the liability and coverage of the researcher and the ordering party (Article 11. §4. 9°).

9)

Any

contributions

and

rules

concerning

the

payment,

repayment

and

compensation to the researchers, participants, and the relevant elements from any agreement between the ordering party and the location (Article 11. §4. 10°).

10) The method of selection of the subjects (Article 11. §4. 11°). 

The application of the articles 11. §4. 1° until 11° in the RILHOU-RCT were explained supra.

In the case of a monocentric trial of phase 1, the CME shall have a period of a maximum of 15 days to give a reasoned advice to the principal investigator. In the case of another trial, the CME shall have a period of a maximum of 28 days to give a reasoned advice to the investigator (Article 11. §5). The competent CME that give the solely advice, can only submit one additional request for extra information to the applicant(Article 11. §6).

43



On 26 August 2014 a request for advice for the RILHOU-RCT was sent to the CME

ZOL

(digital

by

email

and

through

national

registered

mail).

A personal presentation of the study was given (by Smeets H) on 10 September 2014 on the conference of the CME ZOL. On 10 October 2014, a preliminary negative advice from the CME ZOL on the request for advice from the RILHOURCT was offered (by email) to the scientific coordinator and the associate supervisor (in copy) (dated on 7 October 2014). A reply with comments and questions from the CME ZOL were added. These were thoroughly answered primarily by the scientific coordinator and sent by a national registered mail on 4 November 2014 to the CME ZOL. A final positive advice was granted by the CME ZOL on 9 December 2014 (De Cock, 2014).

The composition and conditions to with a CME must to comply (partial or full recognition) are described in detail in article 11/1 and 11/2. Article 4 clarifies this topic (K. B. 4 April 2014 to determination of implementing measures of the law of 7 May 2004 concerning experiments on the human person, in relation to the Ethics Committee B. S. 16 May 2014).

4.3.9 Chapter IX: Admission of the Minister in case of trial This chapter includes article 12 until article 18 and focuses on the ordering party who wishes to organize a (clinical) trial in Belgium. The ordering party shall address its request to the Minister of health by registered mail (Article 12). 

Article 12 until article 18 is not applicable for the RILHOU-RCT because this study belongs to an experiment and not to a (clinical) trial.



Article 2.7° defines 'trial' as follows: "A 'clinical trial' hereinafter referred as 'trial' is an investigation in the human person that is being designed to research the clinical, pharmacological and/or other pharmacodynamic effects of one or more medicinal products to establish or confirm the tolerance, efficacy and safety of a drug, ...".

44

4.3.10

Chapter X: Changes in the implementation of an

experiment.

When the ordering party wishes to make substantial changes to the protocol after the beginning of the experiment that could effect "the safety of the subjects or lead to a different interpretation of the scientific documents that substantiate the course of the experiment, or if these changes are significantly different", the researcher has to report in an amendment the reason and content of these changes to the CME. In case of a trial the ordering party notifies the Minister of health by a registered letter (Article 19. §1). With a monocentric experiment the CME analyzes this request to amend, in accordance with article 11. In the case of a multicenter experiment it’s the CME which is legally valid to reported an advice (Article 19. §2). 

There were no substantial changes made to the protocol by the ordering party after the approval of the RILHOU-RCT by the CME ZOL.



Minimal changes to the protocol after final approval by the CME ZOL (deviations) were made before the commencement of the experimental study. This led to minor adjustments in the RILHOU-RCT DRAAIMAP + FMEA. Adjustments were reported to the scientific coordinator, which gave his approval. The minimal changes did not affect

the

protocol

so

that

there

could

be

no

question

of

protocol

deviation/violations.

4.3.11

Chapter XI: End of the experiment

In the event of a monocentric or multicenter experiment the ordering party informs within 90 days of the end of the experiment the competent CME about the end of the experiment (Article 21. §1). In case the experiment is stopped at an earlier stage than expected, the time limit is reduced from 90 days to 15 days. The reasons for early termination should be clearly described (Article 21. § 2). 

Since VZW Health does not act as 'a classic ordering party’, the competent CME was being informed by the scientific coordinator (De Cock, 2014). Since the death of De Cock Johan († 6 December 2016) the study coordinator (and contact person RILHOU-RCT) will accomplish further tasks.

45

 On 30 November 2015 a petition request was transferred to the CME ZOL by the scientific coordinator for an extension period for the RILHOU-RCT. An approval to extend the RILHOU-RCT was obtained until 31 December 2016.  On 7 December 2015, an extension of insurance policy no. 45.326.306 was granted by Ethias for the acting validity period of 1 January 2016 until 31 December 2016.

4.3.12

Chapter XII: Suspension or prohibition of the

experiment and violations In this chapter the law of 7 May 2004 describes the suspension, prohibition of the experiment and the violations. The CME informs the ordering party and principal investigator who now have a week the time to assess their advice. When there is an imminent danger the period of one week can be shortened (Article 22. §1). If, after receival of the advice of the ordering party/investigator, the CME still considers that the conditions to perform the experiment are violated, the Minister of Health will be informed by the CME (Article 2. 2°). The Minister of Health may suspend or prohibit the experiment. The suspension or the prohibition is legally operative as soon that the ordering party is informed (Article 22. § 2). If there are objective grounds to assume that the sponsor, investigator or any other person involved in the experiment no longer can fulfil their obligations, the Minister of Health informs immediately on his or her own initiative (or on a proposal from the CME) the appropriate person(s). A code of behavior will be given by the Minister of Health and must be followed to correct the situation (article 23. § 1).

4.3.13

Chapter XIV: Review of the agreement with good

clinical practice and good manufacturing practice of investigational medicinal products This chapter describes article 26 (divided into 5 paragraphs). Article 26. §5 states that the detailed guidelines concerning the documentation related to the trial (the permanent file of the trial), archiving methods, qualification of the inspectors and inspection procedures for the review of the conformity of the trial with this law shall be ascertained by the King. Directive 2001/20/EG (No. 14) mentions that ‘non-commercial clinical trials’ conducted by researchers without the participation of pharmaceutical

46

industry can lead to great benefit for the involved patients. The directive takes into account the individuality of trials whose design requires no specific production methods or packaging. For the purposes of this directive, article 15 paragraph 5 referred to the archiving methods that are recorded and reviewed in accordance to the procedure as meant in article 21 paragraph 2. 

The ICH-GCP guidelines were applied for the design and implementation of the RILHOU-RCT, as well as the latest version of the Declaration of Helsinki (De Cock, 2014).

4.3.14

Chapter XV: Reporting of adverse events

The law defines "adverse event" as follows: “a harmful occurrence at a patient or subject in the treatment group during an experiment, which is not necessary in connection with the treatment" (Article 2, 9°). All serious adverse events must be immediately reported to the ordering party (sponsor) by the investigator. When it comes to a serious adverse event in accordance with the protocol or investigation file there is no immediate reporting to the ordering party required. The immediate reporting is followed by detailed written reports with the code numbers of the subjects (Article 27. §1). The ordering party shall keep detailed records of all reported adverse events by the investigator or investigators. At the request of the ordering party all the registers with adverse events are submitted to the Minister of Health, when the experiment or trial is carried out in Belgium (Article 27. §4). 

Serious adverse events are reported to the scientific coordinator.



In the investigation file of RILHOU-RCT the observation and reporting of possible adverse events was taken into account, before the start of the study. Each file has 3 pages registration ‘adverse events’ (De Cock, 2014). At the end of the study adverse events were passed to the scientific coordinator. In case of occurrence a detailed register of all adverse events/side-effects in the RILHOU-RCT is transferred to the Minister of Health by the scientific coordinator.



In this study no serious adverse events/side effects occurred, hence were reported. Adverse events such as skin irritations caused by the ECG/ICG electrodes arose but do not count as adverse events that can be attributed to the experiment.

47

4.3.15

Chapter XVI: Reporting of serious side effects

The law makes a distinction between side effects and unexpected side effects with regard to drugs (Article 2. 5° and article 2. 6°). Relevant information about suspected unexpected serious adverse reactions that have led (or could lead) to the death of a subject are registered by the ordering party. In case of a clinical trial, the ordering party has to report it within seven days to the Minister of Health; the competent authorities of all the concerned Member States and to the competent CME. Relevant information about the consequences have to be communicated within a further period of 8 days (Article 28. §1, B. S. 18 May 2004). 

The RILHOU-RCT does not use drugs. An explanation of adverse events in the file was supra mentioned in 4.3.2. Chapter II: Definitions and application.

4.3.16

Chapter XVII: Liability and insurance

The ordering party is faultless liable for the damage that the participant or in case of death the entitled party has suffered. The damage that the participant sustained can have a directly or indirect link with the experiment. Any contractual provision to limit this liability and responsibility will be considered null and void (Article 29. §1). Before the experiment is started the ordering party should take an insurance that covers this liability, the same for each person intervening in the experiment. This regardless of any possible connection that could exist between the intermediary, the ordering party and participant. The ordering party or legal representative must be located in the European Union (Article 29. §2). In Belgium, the subjects or the entitled party can summons the insurer directly for the judge of the resort where the fact has occurred from which the damage had occurred. This both for the judge of the place of residence of the participant as well for the judge of the jurisdiction (resort) where the seat of the insurer is located. The insurer can’t oppose nor object defense against articles which derive from the law or from the insurance contract, relating to the participant or its entitled party (Article 29. §3).

• Application of this article was described in 4.3.2 Chapter II: Definitions and application.

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4.3.17

Chapter XIX: Special provisions for non-commercial

experiments After advice of the Advisory Committee on Bio-ethics the King can recognize institutions that have submitted a request where the purpose is mainly focused on research without profit. When the trial is recognition as a 'non-commercial experiment' it must comply to the content as described in article 2. 15 ° (article 31. § 1). The ordering party in the event of a 'non-commercial experiment’ is exempted from contribution and retribution (Article 30 and 31. §5). 

The RILHOU-RCT is recognized as “non-commercial experiment” (Article 2. 15°).

This makes that the ordering party “VZW Health” is exempted from the contribution and retribution, as described in article 30 and article 31. §5.

4.3.18

Chapter XX: General

Chapter XX describes articles 32 to 33bis. These articles explain general guidelines and mention that no one should participate at the same time to different biomedical trials of Phase 1 (Article 32. §1). Before an experiment may startup, the researcher is committed to consult the database (a registry set up for healthy volunteers who lend themselves to an experiment) (Article 32. §2, §3). Who commits an infringement on the following articles: 5 to 10; 12; 17; 19 to 21; 22. §2; 24; 25; 27; 28. §1 and §2, 29. §2 and 32. §1 will be punished with a prison sentence of one month until two years, and/or with a fine of 500 euro until 250.000 euro (Article 33. §1). These punishments apply to penalties provided in other laws, and the application of disciplinary measures. Article 33. §3 states that natural and legal persons are responsible for the fines and the costs arising from the sentences that were pronounced against their organs or representatives. 

As mentioned earlier, the RILHOU-RCT consists of seven studies. When a subject is participates in one of these studies, the subject is no longer included in another study of the RILHOU-RCT.



In this study none of the participating subjects were registered or participating in a biomedical Phase 1 Trial.



From the ethical point of view, students associated to FICO were not selected to participate in the study.

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4.3.19

Pro memorie

The law of 7 May 2004 (concerning experiments on the human person) describes another 5 chapters that were not elaborated above, because these are irrelevant to the RILHOU-RCT. 

Chapter IX/1: article 18/1 ‘till 18/3 describes the legislation on experiments where human body material is obtained, processed, stored and/or made available.



Chapter X/1: article 20/1 (divided into 4 paragraphs) refers to the advice of the Committee for drugs determined for human use in case of trial, and the enforcement of this law.



Chapter XIII: article 24 (divided into 7 paragraphs) describes the rules of the manufacturing, import and distribution of investigational medicinal drug products.



Chapter XIIIbis: article 25 describes the legislation for research on labelling of medicinal products.

5 Part II: Heart Rate Variability (HRV) 5.1 Introduction: the heart The osteopathic founder Andrew Taylor Still (1828-1917) described in his book "The Philosophy and Mechanical Principles of Osteopathy" that "the heart" is the mother of all nerves, which possesses wisdom and enhances life force. The heart has an important function in the association of the material body, spiritual body and the essence of the spirit (Still, 1902). Throughout history, the heart is in many cultures considered as the source of emotions, passion, wisdom (Salem, 2009; Salem, 2007; Gossman et al 2016) and intuition (McCraty et al. 2004a,b). Still also recognized that electric and magnetic forces in the body are important for optimizing and maintaining good health (Still, 1902).

The heart generates an electromagnetic field which electric component is 60 times greater than in amplitude and were the magnetic component is 5000 times stronger compared to the electromagnetic field of the brain (McCraty et al. 2004c). Anatomically the heart consist of a helix ventricular myocardial band (Kocica et al. 2007; Carreras et al. 2006) that acts as a peristaltic dynamic suction pump (Forouhar et al. 2006;

50

Carreras et al. 2006). It provides that the blood can circulate in his own biological momentum like a spiral throughout the body (Hegde, 2008; Marinelli et al. 1996). The heart belongs to an endocrine organ that produces cardiokines (Clerico et al. 2006; Ogawa

and

the

Bold,

2014)

and

exchanges

information

through

different

communication pathways with various organs (Nakamura and Sadoshima, 2014), cells and the universe (Lipton, 2015; Pearsall, 1999; Pert and Chopra, 1999; Pert, 2007).

5.2 Communication pathways of the heart There are several bi-directional communication pathways between the heart, the brain and other organs/ tissue such as: neurological communication (CNS, ANS) (Ruffini et al. 2015), bio-physiological communication (Ponard et al. 2007; Cavallari et al. 2010), biochemical communication (Ligands) (Geletta et al. 2007) and bio-energetic communication (Rosch, 2009; McCraty, 2004; McCraty et al. 2004a,b,c). Most of the communication mechanisms were already described in detail in Osteopathic dissertations and articles (Franssens, 2005; Vander Mast, 2008; Henley et al. 2008; Minderhout, 2009; Carrillo, 2013; Giles et al. 2013; Girsberger et al. 2014; Carmans, 2015; Janssens, 2015; Buschmann, 2017).

In this dissertation some additions on different communicational pathways are being further elaborated.

5.2.1 Neurological communication The relationship between the brain and the heart forms a basis for the use of HRV in the psychophysiology (Brentson et al. 1997). Neurons of the vagus nerve are situated more in the nucleus ambiguous and less in the vagal nucleus (Kukanova and Mravec, 2006). According to McCraty and Childre, the vagus nerve contains 85 to 90% of visceral afferent fibers (McCraty and Childre, 2010). Neural axons in the ganglia nodosum are sending through the vagus nerve sensory information to the nucleus tractus solitarius. Through this nucleus a direct connection arises towards the lymbic system (amygdale, hypothalamus, thalamus) (McCraty, 2015; McCraty and Childre, 2010) and prefrontal cortex (Park and Tallon-Baudry, 2014; Taylor et al, 2010; McCraty et al. 2009; Thayer, 2007) (Figure 1). According to recent evidence there is a direct afferent pathway from the dorsal vagal complex to the (pre) frontal cortex (Klarer et al.

2014; McCraty et al. 2009). Several authors have stated that the cardiac afferent nerves affect the homeostasis and modulation of the activity in the subcortical and

51

cortical brain areas. Both brain areas are involved in the processing of perceptual and emotional processes (McCraty and Zayas, 2014; McCraty et al. 2004a; Wager et al.

2009). The innervation system responds as a kind of antenna that is tuned towards and responds to magnetic fields, produced by the hearts of other people (McCraty et al.

1998).

Figure 1: Afferent pathways of the nucleus tractus solitarius to the subcortical Levels (McCraty, 2015)

The heart is a sensory organ that has an information coding system and a processing center (McCraty et al. 2004a; Armour and Kember, 2004; Pearsall, 1999) and is connected with a complex intrinsic cardiac nerve system (Figure 2) (Kukanova and Mravec, 2006). Therefore the heart would be able to function independently of the cranial brain. The heart would contain a possibility to learn and remember, even for functional decisions (McCraty et al. 2004a). The intra-cardiac neurons behave independent and have an extensive network that sufficiently can be considered as a private 'little brain' (Armour, 2008).

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Figure 2: Microscopic image of interconnected intrinsic cardiac

Ganglia in the human heart (Shaffer et al. 2014)

5.2.2 Bio-physiological communication (pulse wave) Cognitive performances fluctuate around a rhythm of 10 Hz. The heart has an influence on the synchronization of the cortical activity by means of modulation on the cortical functions by afferent input impulses upon the thalamus neurons. The patterns and stability of the rhythm occur through afferent heart inputs, instead of the amount of neural discharges within the cardiac cycle. These afferent heart inputs are important in the modulation of the thalamus activity and have encompassing consequences for the brain function (McCraty and Shaffer, 2015).

5.2.3 Bio-chemical communication The heart belongs to an endocrine organ that produces and secretes more than thirtyfive different cardiokines (Addendum 4). The most famous cardiokines are: Atrial Natriuretic Peptide (ANP or ANF), Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP) (Tota et al. 2010). Some cardiokines have an effect on HRV (Addendum 5). Atrial cardiomyocytes produce mostly ANP (Figure 3) and produce urodilatin, while BNP especially are produced by the ventricular cardiomyocytes. The production of CNP is mainly produced by the endothelial cells and cardiac fibers (Clerico et al. 2011; Ogawa and the Bold, 2014). Intrinsic cardiac adrenergic cells synthesize and secrete catecholamines such as dopamine, norepinephrine and epinephrine (VĂŠgh et al 2016; Lehmann et al. 2013). New insights support the

53

physiological relevance that oxytocin also is produced by the heart (Gutkowska et al.

2014; Gutkowska et al. 2000).

Figure 3: Metabolic communication between cardiokines ANF/ BNP and other Tissues (Nakamura and Sadoshima, 2014)

The endocrine heart serves as a key role in the regulation of cardiovascular and renal systems (Figure 4); adipose tissue function and growth; lipolytic effects in human fat cells. There is a mutual relationship between cardiac natriuretic hormones (CNHs) and adipokines (metabolic disorders), body fat distribution, gonadal function, steroid hormones, circulation (Clerico et al. 2011), hydro-electrolytic homeostasis, the central and autonomic nervous system, kidneys, adrenal glands, vascular endothelium, adipose tissue and the immune system (Buglioni and Burnett, 2013; Clerico et al.

2006).

54

Figure 4: Effects of cardiokine ANP on the cardiometabolic regulation on blood fibers, kidneys and adipocytes (Buglioni and Burnett, 2013)

5.2.4 Bio-energetic communication Led by ligands (antigens, hormones, neurotransmitters, etc.) (Clerico et al. 2011; Nakamura and Sadoshima, 2014) and electromagnetic fields (Rosch, 2014; Gerber, 2011; Rosch, 2009) there emerges an ‘inter-organ’ and ‘inter-cellular’ communication (Figure 5).

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Figure 5: Receptor activation by ligands and electromagnetic fields (Rosch, 2009)

According to McCraty et al. there would be a 'heart to heart' synchronization between subjects through a 'heart-to-heart bio-communication' (Rosch, 2009). Research shows when a mother focuses with her intention on her baby, a synchronization between the brain waves (EEG) of the mother and the heartbeats (ECG) of her baby occurs without any physical contact between the mother and her baby (Figure 6). An inverse relationship between the synchronization of the EEG from the baby and ECG of the mother was not found (McCraty and Shaffer, 2015).

Figure 6: Synchronization between the brain waves of a mother (EEG) and the ECG of her baby (McCraty and Shaffer, 2015)

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Some researchers suggest that the physical heart is linked to an information field that is not linked to the classical limits of time and space. The heart receives and processes information on a future event, before the event actually occurred (McCraty et al.

2004a,b). Backsters Cleve studied the biocommunication in plants, animals and human cells (Solfvin, 2009). His research results show that there is a continuous extrasensory communication between all living organisms, with each other and their environment.

Organisms have the ability to reject on the environment and respond to this primarily (Backsters, 2013). Van Wijk et al examined the influence of 'Healing intentionsâ&#x20AC;&#x2122; on simple algae (Acetabularia acetabulum) and discovered a strong change during the healing session on the photon count distributions. The authors suggest that during a â&#x20AC;&#x2DC;healingâ&#x20AC;&#x2122; a shift occurs in the cyclical components of the photon emission (Van Wijk and Van Wijk, sd; Van Wijk and Van Wijk, 2003). Researchers such as Backster, Popp and Korotkov have shown implications on the effect of intention. The slightest thought seems to have influence on the bio light from someone else, both strengthening as weakening (McTaggart, 2013).

5.3 Heart rate variability Heart Rate Variability is a statistical measurement method based on the R-R intervals of the heart rhythm. The scientific relevance of HRV is shown, for instance in the PubMed database, where between 1980 and 2013 were more than 10 000 abstracts published with 'heart rate variability' in the title or abstract (Sassi al, 2015). Scientific research with HRV is also used within alternative medicine (CAM) (Mishra et al. 2011; Wang et al. 2012; Cantalino et al. 2014; Franssens, 2005; Vander Mast, 2008; Henley et al. 2008; Minderhout, 2009; Giles et al. 2013; Girsberger et al. 2014; Carmans, 2015; Janssens F, 2015; White, 2012).

Osteopathy belongs under CAM and is a form of a drug-free, non-invasive manual medicine (Ruffini et al. 2015). Sine 1999 Osteopathy is in Belgium classified under the exercise of non-conventional medicines (Law 29 April 1999, concerning the nonconventional practices...). The osteopathic concept consists of an integration of six models: the biomechanical-structural model, the respiratory-circular model, the metabolic-nutritional model, the neurological model, the behavioral psychosocial model and the bio-energetic model (Hendryx, 2014).

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5.3.1 Definition of heart rate variability HRV has become a conventionally accepted term to describe variations of both instantaneous heart rates as RR-intervals (Camm et al. 1996). In the ECG wave component R of the QRS complex corresponds with a left ventricular depolarization (Freeman, 2006). In order to achieve HRV the time intervals between consecutive heart beats (R-R) are analyzed (Figure 7).

Figure 7: Wave components PQRST in a normal ECG (U wave is not shown in the Figure) (Reed, 2005. At Karim et al. 2011)

5.3.2 Background The first written description of the heart rate "Measured by the pulse" was found in the writings of Herophilos (ca. 335-280 BC). As a physician, anatomist and scientist, he was the first to divide blood vessels in arteries and veins. He observed the function and physiology of the cardiovascular system and came to the conclusion that there was a difference between arteries and veins, such as the rhythmic pulsing of the artery.

Herophilos was probably the first person that has measured the heart rate (Billman, 2011). The astronomer Johannes Kepler (1571-1630) would have recorded the first heartbeat (Bedford, 1951). In 1733 Hales observed the permanent variations of the heart rate and arterial blood pressure. In horses he found a relationship between the breathing and heart rate, as well as between the blood pressure (Brentson et al. 1997).

At the end of the 19th and beginning of the 20th century Willem Einthoven used the galvanometer for accurate measurement of changes in the electrical conduction

58

system of the heart. In this period, the first continuous recording of the electrical activity of the heart was registered (Billman, 2011). Einthoven introduced the name electrocardiogram and named the wave components PQRST, something which in the past was referred to as ABCD (Meijler, 1987). In 1868, Franciscus Donders examined the relationship between breathing with the vagus nerve and RSA. Hamlin et al. (1972) showed that RSA with dogs and other mammalian species was the result of vagal activity (Billman, 2011). Traube speculated that the heart rate was controlled in certain physiological nuclei of the brainstem through direct irradiates from the respiratory medulaire center (Brentson et al. 1997). Cannon showed that with a "strong arousal" an animal would mobilizes itself with the response of "fight or flights". This response would be caused by a combination of increased sympathetic activity with the hormone adrenaline (Brown and Fee, 2002). In 1915, Cannon reported his first findings in his book “Bodily changes in pain, hunger, fear and rage" (Cannon, 1915). He concentrated on the complexity of the chemical neurotransmission, homeostasis (a term coined by Cannon in 1926) and psychosomatic medicine (Brown and Fee, 2002). Adrian et al.

(1932) published the first recording of sympathetic activity in anesthetized cats and rabbits (Barman, 2009). Hon and Lee showed the first clinical relevance for HRV in 1963. They used HRV to investigate fetal stress (Brentson et al. 1997). Around this time the functional relationship between the heart and the brain was also investigated (Aydin et al. 2011). Around 1980, researchers analyzed alongside the time domain the frequency domain through the short-term HRV analysis (Akselrod et al. 1981) and nonlinear analyses (Goldberger, 1996).

Since 1996, standard guidelines for the determination of HRV were described in a report of ‘The Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology' with the purpose to achieve a uniformity in order to be able to compare HRV analysis (Malik et al. 1996; Malik, 1996; Brentson et al. 1997). In 2015, a document with respect to the evolution of new methods in physiological and clinical studies was released by ‘The e-Cardiology ESC Working Group and the European Heart Rhythm Association co-endorsed by the Asia Pacific Heart Rhythm Society’ in addition to the report of the Task Force (1996) (Sassi et al. 2015).

Despite the standard guidelines of the Task Force to obtain HRV these guidelines are not always applied (Nunan et al. 2010).

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5.3.3 General HRV can be considered as an intermediate effect measure for assessing the function of the ANS (Pinna et al. 2007). Various HRV parameters can be used for quantifying sympathetic and parasympathetic activity (Sassi et al. 2015). By clustering the HRV parameters information can be obtained about: the total variability (Gernot, 2014), parasympathetic activity (Acharya et al. 2006; Kleiger et al. 2005), sympathetic activity, the sympathovagal balance (Connes, 2010), the scaling behavior of the ANS and the cardiac complexity (ChuDuc et al. 2013).

Farah et al. investigated the intra-individual and inter-individual variability (biological

variability) and intra-observer reliability of the short-term heart rate variability in adults.

One conclusion was that HRV parameters (time domain, frequency domain) both are reliable in adults, but that the time domain is more reliable compared to the frequency domain. Better reliability indicators were observed when HRV parameters were analyzed by the same observer (Farah et al. 2016). Heart rate variability can be affected by several external and internal variables (Addendum 5 and 6).

HRV can serve as a predictor for the assessment of health status (Thielmann and Boeckelmann, 2015; Kemp et al. 2012; Karemaker and Lam, 2000). Changes in HRV patterns can be an early and sensitive indicator of health problems. Itâ&#x20AC;&#x2122;s being assumed that the beat-to-beat variations in the heart rhythm is an indirect measurement for cardiac health. These are being determined by a good balance between the sympathetic and parasympathetic activity. The balance in the beat-to-beat fluctuations is mainly determined by the parasympathetic, because this gives the fastest effect. A high variation in the heart rate is a sign of a good adaptation capacity, what emphasizes a good health characteristic with a good functioning autonomous control system.

A reduced HRV is often an indication of abnormal and insufficient adaptation of the autonomous ANS; the physiological function has been affected. However, exact diagnoses can only be made after further research (Brentson et al. 1997; Pumprla et al.

2002; Sztajzel, 2004). A reduction of HRV is being noticed in various groups of pathologies such as: cardiovascular diseases (Hillebrand et al. 2013; Thayer et al.

2010), respiratory diseases (Reis et al. 2010; Takabatake et al. 2001; Roque et al.

2014), neurological disorders (Tombul et al. 2011; Alonso et al. 2015; Conte et al.

2015; Devos et al. 2003; EvrengĂźl et al 2005; Degiorgio, et al. 2010), gastrointestinal disorders (Tougas, 2000; Mazurak et al. 2012; Tillisch et al. 2005), kidney disease

60

(Ranpuria et al. 2008; Morris et al. 2007); immunological disorders (Przybylska-Felus et al 2014; Evrengül et al. 2004; Tumiati et al. 2000), hematological Disorders (Svenungsson et al. 2001; Poręba et al. 2014), postmenopausal osteoporosis (Tosun et al. 2011), metabolic disorders (Raimundo and Godleski, 2015; Peters et al. 2015), stress (Thayer et al. 2012; Hjortskov et al. 2004; Vrijkotte et al. 2000), epilepsy (Evrengül et al. 2005); Psychiatric conditions (Shah and Vaccarino, 2015; Gorman and Sloan, 2000; Henry et al. 2009; Shinba et al. 2008; Valkonen-Korhonen et al. 2003; Licht et al. 2008; Yang et al. 2010).

HRV analyses can be divided in three domains (Table 1) (Karim et al. 2011).

Table 1: HRV parameters in different domains (Karim et al. 2011) Time-Domain Mean RR (ms) STD RR (SDNN) (ms) Mean HR (bpm) STD HR (bpm) Min HR (bpm) Max HR (bpm) RMSSD (ms) NN50 (count) pNN50 (%) Geometrische analyse RR triangular index TINN (ms)

Frequency domain

Nonlinear

FFT spectrum and AR Spectrum VLF (0-.04 (Hz) Peak (Hz) VLF(0-.04 Hz) power (ms²) VLF (0-.04 Hz) Power (%) LF (0.04-.15 Hz) Peak (Hz) LF (0.04-.15 Hz) Power (ms²) LF (0.04-.15 Hz) Power (%) LF (0.04-.15 Hz) Power (n. U.) HF (0.15-.4 Hz) Peak (Hz) HF (0.15-.4 Hz) Power (ms²) HF (0.15-.4 Hz) Power (%)

Poincare plot SD1 (ms) SD2 (ms) Recurrence plot Lmean (beats) Lmax (beats) REC (%) DET (%) ShanEn Other ApEnD2

HF (0.15-.4 Hz) Power (n. U.) Totaal Power (ms²) LF/HF Power (ms²)

SampEn DFA: α1 DFA: α2

Our body produces signals, which are usually continuous analog. This means that at any time there is a signal, which has a value. This value itself has a continuous variable so that basically each value is possible. A signal can generally be defined as an information-holding energy form that has to be registerable. In addition to the energy that intrinsically involves a signal (electrical, chemical, mechanical, electromagnetic, etc.), signals are subdivided depending on the nature of the process that generates the signal in deterministic and stachostic signals. Deterministic signals are fully defined and can be described by a non-chance dependent function. Stochastic signals are not defined and can only be written with a probability mass function, which makes new values of this signal unpredictable. The measured signal consists of a continuous quantity variable plotted in function of the time. This is visible at an ECG registration where at any time the amplitude has a value with unlimited resolution. Using an analogdigital conversion (AD-conversion), the analog signal is converted into a digital signal wherefore the data can be stored digitally. The sampling frequency and resolution

61

(signal bit) are important. The ‘Nyquist-Shannon sampling’ criterion is used which means that the sampling frequency is at least two times higher compared to the highest frequency in the present signal (Nyquist, 1928; Shannon, 1984). If this condition is not granted a process of aliasing (high frequencies in the analog signal return as low frequencies in the digital signal) will occur. Before the AD-conversion takes place an anti-aliasing filter (low pass filter) is applied to the analog signals (Van Putten and Kees, sd).

6 Part III: The VU-AMS device In 1984, Lorenz van Doornen developed the HRM (Heart rate monitor), which is one of the precursors* of the current VU-AMS version 5fs (Figure 8). This version is a noninvasive ambulatory monitoring system developed by the Department of Biological Psychology and the Technical Department of the Faculty of Psychology and Education of the Free University of Amsterdam. The device is specifically designed for the ambulatory measurement and registering of an ECG/ICG and the electrodermal activity (EDA). The VU-AMS is purposed in several psycho-physiological laboratories and is used mainly for ambulatory research. An advantage of ambulatory research is that psycho-physiological parameters aren’t affected by an artificial laboratory situation. At the Department of Biology and Psychology at the Free University of Amsterdam and the Dutch Twins Registry the VU-AMS performs a key-function in genetic research in the field of stress-reactivity on the ANS and cardio vascularization (Cybulski G, 2011; Lien and the Geus, 2014).

* HRM, HAM, AMD01-AMD03, AMS 3.1-AMS 3.6 (with SCL), AMS 4.0-4.6 (ICG) before settling on VU-AMS [version]. The current version of the VU-AMS device 5fs.

Figure 8: The VU-AMS device 5fs

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6.1 VU-AMS and publications The VU-AMS device is used worldwide by more than 45 research groups. More than 100 publications (with a variety of investigations) were already provided with the VUAMS device, for example: the effect of acute psychological, social and biological stress (Generaal et al 2016; Paine et al. 2013; Roelofs et al. 2007); in psychological distress and physiological reactivity (Busscher et al. 2015); ADHD (Luman et al. 2008a,b, Luman et al. 2007); aggression (Scarpa et al. 2008); anxiety and depressive disorders (Busscher et al. 2013; Hovland et al. 2012, Verhoeven et al. 2011; Penninx et al. 2008; Penninx, 2007), burnout (Van Doornen et al. 2009); the effect of alcohol on the HPA axis and on the ANS (Boschloo et al. 2011); sleep disorders (Hovland et al. 2013); migraine (Schoonman et al. 2007); chronic work stress (Vrijkotte et al. 2000); the relationship between ANS with metabolic and immunological risk factors (Nederhof. et al. 2015; Vrijkotte et al. 2015); the circadian rhythm (Van Eekelen et al. 2004), genetic research (Riese et al. 2014; Out et al. 2010); vagal activity (Kogan et al. 2014); the relationship between mother and child on the ANS (Braeken et al. 2013; Gademan et al. 2013; Van Dijk et al. 2013). On the website of the VU-AMS different publications can be consulted overlapping the period 1995 - 2017 (At: http://www. Vu-ams.

Nl/research/publications/).

6.2 The electrocardiogram (ECG) The electrical activity of the heart muscle is registered through an ECG and displayed in an electrocardiogram (Figure 9). There are different methods to obtain an ECG, such as extremity distractions and chest wall distractions (precordial). In the ECG the P-top displays a representation of the atrial depolarization. On the other hand the QRS complex of the ECG represents the ventricular depolarization. The ST-segment (plateau phase) with the T-top (rapid phase) represents the ventricular repolarization (Dubin, 2011).

63

Figure 9: Electrocardiogram with PQRSTU-wave components (Anaesthesia UK, 2004)

The VU-AMS uses bipolar extremity derivations by means of 3 electrodes for recording the electrocardiogram. The ECG signal is measured and transformed with a 16-bit resolution with a sample frequency of 1000 Hz.

The first ECG electrode (V-) is placed just below the right clavicle, four cm lateral to the sternum. The second electrode (GND) is placed right between ribs 11 and 12. The third ECG electrode (V+) is being attached on the apex of the heart on the left lateral side of the ninth rib, near the processus xiphoideus. On figure 10 is the third ECG Electrode marked with number 3, but this is too low. The black arrow refers that number 3 should be placed slightly higher, at the level of the processus xiphoideus.

6.3 Impedance cardiography (ICG) ICG measures the total electrical conduction across the thorax and the changes in the various cardiodynamic parameters, for instance: stroke volume (SV), heart rate (HR), cardiac output (CO), left ventricular ejection time (LVET), pre ejection period (PEP) (Sima et al. 2015). The ICG reflects the changes in the thorax impedance in response to the respiration and the ejectment of blood from the ventricle into the aorta (van Dijk et al. 2013). Two methods (bipolar and tetrapolair) can be used for the recording of ICG. A bipolar method uses 2 electrodes. In this method, both the generating component as well as the impedance are merged into one electrode. A tetrapolair method uses 4 electrodes, consisting of two generating electrodes and two impedance

64

electrodes. The application and receiving of the signals are thereby separated (Cybulski, 2011).

The VU-AMS 5fs uses a tetrapolaire method. On the front of the thorax two impedance electrodes are placed. One ICG electrode (V-) is at the notch of the incisura jugularis sternalis (Figure 10: No. 6). The second electrode (V+) is attached on the processus xiphoideus (Figure 10: No. 7). On the back two electric-generating electrodes are attached. One electrode (I-) is placed on the spine, at least 3 cm above the first impedance electrode (V-) (Figure 10: No. 4). The second electrode (I+) is placed on the spine at least 3 cm below the second impedance electrode (V+) (Figure 10: No. 5) (van Dijk et al. 2013). The two generating electrodes on the back deliver a constant electric current flow of 50 kHz (350 µA) through the subject (Cybulski, 2011).

Figure 10: Placement of the ECG and ICG electrodes (VU-AMS)

PEP can be extracted from the ECG and LVET, what represents the contractility of the heart muscle. The time interval is calculated from the beginning of the ECG Q-wave to the B-point of the dZ/dt wave, and is expressed in milliseconds (ms). PEP can serve as a useful measurement of the sympathetic effects on contractility of the heart muscle (van Dijk et al. 2013; Berntson et al. 2008; Goedhart et al. 2008; van Lien et al. 2015).

A reduction in the PEP value (short PEP value) is associated with an increase in sympathetic cardiac control (Licht et al. 2010; Norman et al. 2011).

Impedance cardiography is a valid non-invasive technique to measure different indicators in the field of cardiovascular functions (Parry and McFetridge-Durdle, 2006).

In 1995 de Geus EJ et al. examined the ambulatory measurement of RSA and respiration rate. The ‘Ambulatory Measurement Device version 43’ (AMD43, Free

65

University of Amsterdam) and laboratoire measurements were used for the assessment of RSA and R-R curves. The t-test of the inter- and intra-individual regression equation showed no significant differences between the AMD43 and the laboratory data (de Geus et al. 1995). Vrijkotte et al. compared the derivative RSA with rMSSD and HF power. A significant correlation was found between RMSSD and HF power (between 0.54 and 0.95, median: 0.89) was found. During the morning, the correlation was lower compared to the night. They concluded that rMSSD could serve as an alternative index for vagal activity (Vrijkotte et al. 2000).

The variability of the heart periods can be associated with the respiratory frequency, which is referred as RSA. There are several methods to extract RSA from an ECG (infra: 7.5). RSA can serve as a benchmark for cardiac vagal control (van Dijk et al.

2013) and is related with the emotional regulation capacity (Rottenberg et al. 2002). A reduced RSA is related to weaker physical and mental health. It can be a predictive parameter for insomnia in healthy people (Palesh et al. 2008).

Table 2: Impedance-generated hemodynamic parameters and definitions (Parry and McFetridge-Durdle, 2006)

Hemodynamic variable

Parameter

Definition

Thoracic fluid status

Zâ&#x201A;&#x2019;= Thoracic impedance

Baseline fluid status in chest

Left ventricular function

CO= Cardiac output

Amount of blood ejected from the left ventricle in 1 minute

CI= Cardiac index

Cardiac output divided by body surface area

Preload

SV= Stroke volume

Amount of blood ejected with each beat

Afterload

SVR= Systemic vascular resistance

Amount of resistance that the heart must pump against

Contractility

dZ/dt= impedance changes over time

Reflects the force of ventricular contraction

PEP= pre-ejectie period

LVET= Left ventricular ejection time

Time from ventricular depolarization to ventricular ejection Period of time over which blood is ejected from the left ventricle

66

6.4 Electrodermal activity Electrodermal activity (EDA) presents the sympathetic activity of the ANS. It refers to the variation of the electrical properties of the skin in response to sweat secretion, which represents the sympathetic activity of the ANS (Beedek and Kaernbach, 2010).

EDA is used to measure the psychological and sensory activity (Jabbari et al. 2010).

The human body contains different types of glands, of which most are thermoregulating. Eccrine sweat glands are not thermo-regulating and are innervated sympathetic. The neurotransmitter in the post ganglion synapse of this system is acetylcholine and not noradrenaline (Wilke et al. 2009). In humans, the eccrine sweat glands appear in high concentrations in both hands and feets. The eccrine sweat glands could be conceived on the basis of a resistance network with variable resistance. This explains why EDA is a very good benchmark for the sympathetic activity of the ANS (Cacioppo et al. 2007; Benedek and Kaernbach, 2010).

Figure 11: Topography of the skin (eccrine sweat gland) (wikipedia. Org) A distinction can be made between endosomatic and exosomatic measurement. The endosomatic method measures the electrical skin tension, without using external electrical voltage. The exosomatic method (DC/AC) on the contrary uses a small electric voltage (Roth et al. 2012). The external power is transmitted through skin conductance (SC) electrodes placed on the skin. A distinction can be made between SC and skin resistance, but both have a reciprocal relationship with each other (Velden

67

and Vossel, 1986). The RILHOU-RCT uses SC. From the skin conductance can both the skin conductance level (SCL) (tonic) and the skin conductance response (SCR) (phasic) be deduced. SCL is described as the general conduction level that is present during the absence of external stimuli. Changes in SCL are slow and depending on various aspects such as the psychological state, state of hydration, dryness of the skin and autonomic regulation. Variations in the SCL are mainly associated with ‘arousal’ what is described by some authors as ‘energetic mobilization'. The SCR is an expression of attention allocation and occurs when an organism is being faced with a new stimulus (orientation reaction) (Merckelbach et al. 1995). The conductivity is expressed in microsiemens (µS), the opposite of resistance (Ohm= Ω). In an individual the conductivity can vary between 2 and 20 μS. The baseline is a state of total relaxation. SCL is a clear benchmark of interpretation of arousal (Dawson et al. 2007).

When a response is being caused by an external stimulus there is a phasic SCR. The skin conduction increases fast and strongly. Characteristic for the SCR are the peaks in the SCL line (Boucsein, 2012). For for the recording of the SC, the VU-AMS used an exosomatic measurement, by means of 2 SC Ag-AgCl electrodes. These electrodes can be placed at different positions on the hand (Dawson et al. 2007). For a good conduction "skin conductance electrode Biopac isotonic 4 oz gel" at the position of the SC electrodes is being used.

6.5 VU-AMS measuring variables 

HR/IBI



HRV: Fast Fourier Transform (FFT) analysis: 

SDNN: standard deviations of the NN (R-R) intervals



rMSSD (root mean square of successive differences): the square root of the average of the squares of the successive differences between adjacent NNs



IBI: time between 2 consecutive R-R tops



Power spectrum: 

LF: low frequency (0.04 - 0.15 Hz)



HF: high frequency(0.15 - 0.4 Hz)



RSA (0.1 – 0.4 Hz)



PEP



LVET



Breathing frequency (R-R respiratory rate!)



SV



CO

68



SCL



Skin conductance respons (SCR)



Tri-axial accelerometer (exercise, motility)

7 Part IV: similarities and differences between software programme VU-DAMS (v3.9) and Kubios (v 2.2) in relation to the HRV parameters 7.1 General VU-DAMS v3.9[1] and Kubios v2.2[2] are progressive software programs to perform various analyses on a digital raw ECG signal. Both software programs were developed by a University (Free University of Amsterdam[1]; University of Kuopio East Finland[2]; van Lien and the Geus, 2014a; Tarvainen et al. 2009). Kubios v2.2 is specialized in the performance of various HRV analyses (Jonkman et al. 2013; Tarvainen et al. 2014).

VU-DAMS v3.9 analyzes limited HRV, but performs various detailed analyses to: ICG, respiratory score and EDA (van Dijk et al. 2013; van Lien and the Geus, 2014b; van Lien et al. 2015; Lole et al. 2014). Both software programs are supported by various operating systems: Windows (Windows XP, Windows 7, Windows 8 and 8.1), Mac OSX, Linux (only for Kubios v2.2). Both software programs can be downloaded for free (At: http://www. Vu-ams. Nl/; http://Kubios URv2.2. Uef. Fi:/). The scientific literature cited both software programs for analyzing HRV (Wang et al. 2013; McKune, Sookan and McKune, 2011; Young, 2014; Vrijkotte et al. 2015; Breaken et al. 2013; At: http://www. Vu-ams. Nl/research/publications/).

7.1.1 Computational methods This section explains the temporal statistical HRV parameters with a comparison between both programs. The used HRV parameters for the RILHOU-RCT (this study) will be indicated. The computational methods and explanation of the HRV parameters are based on the original and current guidelines by the Task Force of the European society of cardiology and the North American society of pacing and electrophysiology 1996 (Sassi et al. 2015; Task Force, 1996a,b; Malik et al. 1996).

Details of the software programs can be found under: User's Guide of the VU-DAMS v3.9

(At:

http://www.

Vu-ams.

Nl/fileadmin/user

_

upload/manuals/URVU-

DAMS_manual_v1.3 . Pdf) and Kubios v2.2 (At: http://Kubios), Addendum 7.

69

7.1.2 QRST detection algorithm The raw ECG IBI can be opened in VU-DAMS v3.9 by data type 'extension. 5fs' (van Dijk et al. 2013). Kubios v2.2 can upload the raw ECG signals depending on the data type (*. Acq; *. Edf; *. Gdf; *. Hrm; *. Txt; *. Dat; *. Sdf, *. Ste; *. Mat). By opening the data in Kubios v2.2, an automatic detection of the algorithm (based on the PanTompkins algorithm) is being performed (Pan and Tompkins, 1985; Tarvainen et al.

2014).

The sampling frequency for the interpolation of the R-wave differs between both programs. Kubios v2.2 standard applies a sampling frequency of 2000 Hz (Tarvainen et al. 2014), while VU-DAMS v3.9 applies a sampling frequency that can be manually adjusted: 250 Hz, 500 Hz or 1000 Hz (recommended). Both programs use a cubic spline method with a sampling rate of 4 Hz for the interpolation.

Both programs have an automatic artefact correction provided for the R-R interval series (Figure 12). Itâ&#x20AC;&#x2122;s possible in Kubios v2.2 to choose different levels (none, very low, low, medium, strong, very strong, custum) for an artefact correction on the R-R interval series. In VU-DAMS v3.9 the QRS-detector carries out automatically three separate analyses on the IBI (1: detection and marking of missed/clipping data; 2: detection of the occurrences of all R-peaks; 3: plausibility control of the duration of each IBI in the context of the surrounding IBI's). The default algorithm settings of the QRS-detection can be manually adjusted (van Lien and de Geus, 2014a). The artefact removal value is standardly set on sigma (Ď&#x192;) 3.5 (statistical distribution with respect to the standard deviation).

70

RR interval rejection

NN datasequence

Interpolation + sampling

Nonlinear HRV

RR data editing

Frequency Domain HRV

Artefact identification

Microcomputer digitising

Time Domain HRV

ECGrecording

Figure 12: Processing block diagram of the ECG to HRV parameters

(Task Force, 1996)

7.2 Time domain method and geometric analysis Time domain parameters are calculated directly from the R-R interval time series and are the simplest parameters of the HRV analysis (Yoo and Yi, 2004). In this type of analysis all the QRS complexes are detected in the ECG. Next all the intervals between the normal and successive QRS complexes are being measured. These are the N-N intervals (normal-to-normal interval) or R-R intervals. The QRS complexes are the result of the sinusoidal depolarization (Kleiger et al. 2005; Malik et al. 1996; Pumprla et al. 2002; Sztajzel, 2004). Geometric methods (time domain) are derived and built up of conversions and sequences of the R-R intervals. This analysis is less sensitive to individual R-R interval abnormalities. It is not necessary to filter the ECG on artifacts and so called non-sinus beats, something that is necessary for the other HRV parameters in the time domain (Kleiger et al 2005; Pumprla et al. 2002; Sztajzel, 2004).

71

Table 3: Time domain HRV parameters in VU-DAMS v3.9 and Kubios,

used in the RILHOU-RCT

Software HRV analysis

VU-DAMS¹

KUBIOS ²

RILHOU-RCT ¹

2

Time-Domain

+

+

x

x

Mean RR (ms)

+

+

x

x

SD RR (SDNN) (ms)

+

+

x

x

Mean HR (bpm)

+

+

x

x

SD HR (bpm)

+

+

x

x

Min IBI (ms)

+

-

Max IBI (ms)

+

-

Min HR (bpm)

+

-

Max HR (bpm)

+

-

SDANN

-

-

ASDNN (index)

-

-

rMSSD (ms)

+

+

x

x

NN50 (count)

-

+

pNN50 (%)

-

+

Geometrische analyse

-

+

RR triangular index

-

+

TINN (ms)

-

+

x

+ present; - absent; x used HRV parameters in the RILHOU-RCT

Both software programs perform various HRV analysis in the time domain, with some differences such as NN50 and pNN50 (Kubios v2.2). By neither of the programs SDANN is being analyzed. Only Kubios v2.2 provides geometric analyses (triangular HRV index (RRtri), poincaré plot (see nonlinear analyses), triangular interpolation or NN interval histogram (TINN) (Table 3).

7.2.1 SDNN SDNN is the standard deviation of all the normal QRS distances, in other words all RR intervals for each of the 5-minute segments of the whole recording. It’s a global index for HRV. SDNN might strongly be influenced by the day/night rhythm. The impact is estimated at 30-40% (Gernot, 2014).

72

7.2.2 SDANN SDANN is the standard deviation of the average NN intervals in each of the 5-minute segments of the whole recording. It is a refined version of the SDNN (Kleiger et al 2005).

7.2.3 ASDNN ASDNN (index) is the average of the standard deviation of all NN intervals for all 5minute segments in 24 hours (Buccelletti et al. 2012). The difference between the longest and the shortest RR-distance is the easiest form of analysis in time domain (Sridhar et al. 2010). ASDNN (index) reflects the influence of the autonomic nervous system on heart rate modulation (Task Force, 1996a,b; Stein and Pantazopoulos, 2011).

7.2.4 SD HR SD HR is the standard deviation of all HR samples of 5-minute time interval. The difference between traditional HRV analyses is that the exact instantaneous heart rate is not acquired on each heartbeat. The standard deviation of these points is the basic parameter for short-term measurements (Grogan et al. 2004).

7.2.5 NN50 and pNN50 NN50 are the number of consecutive NN intervals with absolute differences of more than 50 ms. pNN50 represents the percentage of differences between adjacent NN intervals differing less than 50 ms (Yoo and Yi, 2004).

7.2.6 rMSSD rMSSD is the square root of the mean of the sum of the squares of differences between adjacent NN intervals. It calculates the standard deviation of the variations in successive R-R intervals (Pumprla et al. 2002).

rMSSD and pNN50 are short-term variables based on the R-R interval differences.

Both parameters quantify parasympathetic modulation of normal R-R intervals driven by ventilation (Kleiger et al 2005).

73

7.2.7 Detrended time series Detrended time series (DTS) is a variation on the time domain analysis. A running average is taken on the rough R-R data, represented by an interrupted curve. The difference between the two curves is indicated by ri (DTS) and represents the fluctuations, in respect of the local average (Ashkenazy et al. 1998).

7.2.8 RRtri and TINN RRtri (Triangular index) and TINN (triangular interpolation or RR intervals) are calculated on the basis of the density of the normal R-R intervals and are being converted into a histogram. Axis-X represents the length of the R-R intervals, axis-Y represents the frequency with which they occurred (Vanderlei et al. 2010). A histogram is the most used statistical method in which the duration of the R-R intervals are plotted against the total number of R-R intervals in a determined period, for example 24 hours (Stein and Pantazopoulos, 2011). The ECG recording duration to perform geometric statistics must be at least 10 minutes (Hoshi et al. 2013).

7.3 Frequency domain analysis HRV analysis in the frequency domain uses a power spectral density method (PSD).

This method gives information about the power, the variety, distribution and function of the frequencies. PSD analysis can be classified as a non-parametric method (FFT; high processing speed) and a parametric method (smoother spectral components).

This is dependent on: the preselected frequency bands, the post process of the spectrum with an automatic calculation of the low and high frequency power components, and the accurate estimation of the PSD on a small number of samples (assumes that these are stationary) (Vandeput, 2010). The parametric method is not included in Table 4.

Auto regression (AR) method can be used on R-R intervals. FFT analysis has the advantage that itâ&#x20AC;&#x2122;s easy in the implementation while AR analysis gives a better resolution, particularly for short samples (GĂźrĂźler et al. 2014). The heart frequency can be divided in different underlying spectra: high frequency power (HF = 0.15-.40 Hz), low frequency power (LF = 0.04-.15 Hz), very low frequency (VLF = 0.012-.04 Hz), ultra low frequency (ULF = < 0.008 Hz), total power (< 0.4 HZ) and the ratio of the lowpower high frequency (LF/HF ratio) (ChuDuc et al. 2013; McCraty and Shaffer, 2015; Oweis and Al-Tabbaa, 2014). The variation of each underlying frequency (y-axis) is

74

expressed and presented on the x-axis. The bigger the variation of the heart frequency is, the larger the area under the curve will be (Sztajzel, 2004). ULF reflects the circadian rhythm, and is strongly associated with SDANN. VLF is associated with the renin angiotensin system and parasympathetic activity. LF (power, n. U.) represents the activity of the baroreceptors, and a mix of sympathetic and parasympathetic activity (Kleiger et al 2005). Both VLF and LF can be obtained through a short-term recording (Âą 5 minutes). Both are similar to ASDNN (index) (Stein and Pantazopoulos, 2011).

The low frequency power is associated with sympathetic activity, while the high frequency is associated with parasympathetic activity (Acharya et al. 2006), though in some literature some alternative views exist on this matter (Goldstein et al. 2011).

The high frequency band represents also the respiratory sinus arrhythmia (RSA) and is associated with rMSSD and pNN50 (Kleiger et al. 2005; Stein and Pantazopoulos, 2011). LF/HF ratio is the balance between sympathetic and parasympathetic activity, though there is some disagreement about this (Connes, 2010; Billman, 2013).

VU-DAMS v3.9 and Kubios v2.2 perform various HRV analyses in the frequency domain (Table 4). Both software programs use the Welch's periodogram method (FFT) with a standard signal preprocessing setting of R-R interval detrending 'smoothness priors matrix' with as Lambda value 500 (fc= 0.035 Hz). Kubios v2.2 provides an additional possibility 'detrending options' for removing the first to the third order of the linear trend of smoothness priors. An autoregression analysis (AR spectrum) is only available in Kubios v2.2. The frequency bands LF and HF are present in both programs, but Kubios v2.2 analyzes additional VLF. Both programs use the default values for the frequency bands based on the Task Force (1996), but can be adjusted manually. Compared in the frequency domain with VU-DAMS v3.9, Kubios v2.2 analyzes a much more comprehensive HRV analysis. In both programs only the FFT spectrum analyses for LF power ms2; HF power ms2 and LF/HF power ms2 are in accordance.

75

Table 4: Frequency domain HRV parameters in VU-DAMS v3.9 and Kubios, used in the RILHOU-RCT Software Analysis HRV Frequency domain

VU-DAMS

1

Kubios

2

RILHOU-RCT 1

2

+

+

x

x

FFT spectrum

+

+

x

x

ULF (<0.003 HZ)

-

-

VLF (0-.04 (Hz) Peak (Hz)

-

+

VLF(0-.04 Hz) power (ms²)

-

+

VLF (0-.04 Hz) Power (%)

-

+

VLF (0.04 Hz) Power (n. U.)

-

-

LF (0.04-.15 Hz) Peak (Hz)

-

+

LF (0.04-.15 Hz) Power (ms²)

+

+

x

x

LF (0.04-.15 Hz) Power (%)

-

+

LF (0.04-.15 Hz) Power (n. U.)

-

+

HF (0.15-.4 Hz) Peak (Hz)

-

+

HF (0.15-.4 Hz) Power (ms²)

+

+

HF (0.15-.4 Hz) Power (%)

-

+

HF (0.15-.4 Hz) Power (n. U.)

-

+

x

Totaal Power (ms²)

-

+

x

LF/HF Power (ms²)

+

+

AR Spectrum

-

+

ULF (<0.003 HZ)

-

-

VLF (0-.04 (Hz) Peak (Hz)

-

-

VLF(0-.04 Hz) power (ms²)

-

+

VLF (0-.04 Hz) Power (%)

-

+

VLF (0.04 Hz) Power (n. U.)

-

+

LF (0.04-.15 Hz) Peak (Hz)

-

+

LF (0.04-.15 Hz) Power (ms²)

-

+

LF (0.04-.15 Hz) Power (%)

-

+

LF (0.04-.15 Hz) Power (n. U.)

-

+

HF (0.15-.4 Hz) Peak (Hz)

-

+

HF (0.15-.4 Hz) Power (ms²)

-

+

HF (0.15-.4 Hz) Power (%)

-

+

HF (0.15-.4 Hz) Power (n. U.)

-

+

x

Totaal Power (ms²)

-

+

x

LF/HF Power (ms²)

-

+

x

x

x

x

x

x x

x

x

x

+ present; - absent; x HRV parameters used in the RILHOU-RCT

76

7.4 Nonlinear analysis There is being insinuated that mechanisms that are involved in cardiovascular regulation interact with other organs and hereafter exhibit a repercussion. Heart rate fluctuations are subordinate to the control of the ANS (Kunz et al. 2012). In natural systems this pattern is characteristic chaotic and not linear. Chaos theories and fractals (nonlinear analysis) are taken into account when these natural caprices and notperiodicity of patterns (Huikuri et al. 2003) are considerated. The dynamic behavior of a system can be described by the state space or phase space. Each state of the system is represented by a point in the space of a multidimensional phase space (Uzun et al.

2001). Gonçalves H et al. examined the HRV of male and female fetuses on the basis of linear and nonlinear analysis. The linear analysis showed an increase of HRV at the male fetuses, compared to the female fetuses. These results were in contrast with the nonlinear analysis where the female fetuses showed an increase of HRV, compared to the male fetuses (Gonçalves et al. 2007).

Nonlinear HRV analyses give additional information about the nonlinear dynamics (specifically about the direction of the behavior and the complexity) of the ANS. Linear analyses exhibit only a similar behaviour between different times periods. Nonlinear analysis can be divided into three groups: poincaré plot, recurrence plot and other (Table 5).

7.4.1 The Poincaré method The Poincaré method expels the time between two following R-tops in function of the time between the previous two R-tops of the ECG. This analysis is a simple quantitative visual technique compared to the conventional Fast Fourier Transform indices. Hoshi et al. have proved that there is a high correlation between SD1/SD2 and Detrended Fluctuation Analysis (DFA) α1. It is suggested that these proportions can be used as a surrogate variable (Hoshi et al. 2013). In healthy people, this yields an elliptical graphical model (Kleiger et al 2005). SD1 represents the width of the ellipse and reflects the fast beat-to-beat (short-term) variability (Acharya et al. 2006).

SD1 is a marker for vagal activity (Singh and Bharti, 2015). SD2 represents the length of the ellipse and reflects the long-term variability (Acharya et al. 2006; Muralikrishnan et al. 2013). The length is affected by the activity of both branches of the autonomic nervous system (Singh and Bharti, 2015). A small ellipse indicates constant R-R intervals (Jia et al. 2012).

77

7.4.2 Recurrenc plot Recurrence plot is a repetition of the fundamental properties of a dynamic system.

This plot visualizes the recurrence behavior of the phase space trajectory of dynamical systems (Marwan et al. 2007).

7.4.3 Entropy Entropy means general a measure of unpredictability of the state, or equivalently of its average information content.

7.4.4 Shannon Entropy Shannon Entropy (ShanEn) is used as index and describes the shape of the distributing of patterns. A great partition is featured with a flat distribution (all patterns are identically distributed and the series carry a maximum amount of information). A small partition is probably to be featured with a subset of patterns, while others are missing or less occurrent (Kunz et al. 2012).

7.4.5 Approximate Entropy Approximate Entropy (ApEn) is a nonlinear complex index for quantifying the regularity of physiological time series (Perakakis et al. 2010). A low ApEn value is associated with a low HRV, and many similar looking patterns or regularity (Pincus et al. 1991).

7.4.6 Sample Entropy Sample Entropy (SampEn) is almost similar as ApEn with the only difference that SampEn distance vector x (i) is not included in the calculation. SampEn is therefore more precise compared with ApEn (Bernardes et al. 2008; Richman and Moorman, 2000).

7.4.7 Detrended Fluctuation Analysis Detrended Fluctuation Analysis (DFA) is used for quantifying the fractal scaling properties for the short (Îą1) and long (Îą2) term interval signals. It is a modification of the

78

root-mean-square (rMSSD) (Acharya et al. 2006). α1 is generally made of 1000 R-R intervals (normal value α1= 1.05), while α2 is based on 12 to 20 R-R intervals (Kleiger et al 2005).

7.4.8 Correlation Dimension Analysis Correlation Dimension Analysis (D2) measures the degree of cardiac complexity of the system that is generated by the R-R interval series. Higher D2 values are associated with a large cardiac complexity of the system (suppression of sympathetic activity). Lower D2 values are associated with a smaller cardiac complexity of the system (suppression of parasympathetic activity) (Bolea et al. 2014). Lower D2 values together with lower HRV are being observed in sick people, compared with healthy people. In healthy individuals D2, ApEn and the circadian rhythm are identified with lower values in the morning and higher values at night. Healthy women exhibit larger D2 values, compared to healthy men (Carvajal et al. 2002). Only the software program Kubios v2.2 provides a nonlinear analysis on HRV (Table 5).

Table 5: Nonlinear HRV parameters in VU-DAMS v3.9 and Kubios, used

in the RILHOU-RCT Software Nonlinear

VU-DAMS

1

Kubios

2

RILHOU-RCT 2

Poincare plot

-

+

x

SD1 (ms)

-

+

x

SD2 (ms)

-

+

x

Recurrence plot

-

+

x

Lmean (beats)

-

+

Lmax (beats)

-

+

REC (%)

-

+

DET (%)

-

+

ShanEn

-

+

x

Other

-

+

x

ApEn

-

+

x

SampEn

-

+

x

DFA: α1

-

+

x

DFA: α2

-

+

x

D2 + x Lmean: Mean line length of diagonal Lines in recurrence plot (RP); Lmax: Maximum line length of diagonal Lines in recurrence plot; REC: Recurrence rate (percentage of recurrence points which form diagonal Lines in recurrence plot; DET: Determinism (percentage of recurrence points which form diagonal Lines in recurrence plot; ShanEN: Shannon entropy of diagonal line lengths probability distribution; + present; - absent; x HRV parameters used in the RILHOU-RCT

79

7.5 Respiratory sinus arrhythmia extracted from the ECG Respiratory sinus arrhythmia (RSA) is the most marked periodic component of HRV. According to the author the range of the respiratory frequency band varies between 0.15 and 0.5 Hz (Brentson et al. 1997; Widjaja et al. 2010; Boyle et al. 2009). This high frequency component is almost exclusively mediated by parasympathetic activity (Tarvainen et al. 2014).

RSA is modulated from the heart rate under the impulse of breathing. Spontaneous respiration is triggered by cardiac activity (Porges, 2011). The relationship between breathing

and

heartbeat

is

bidirectional.

RSA

and

heart

rate

reflect

the

cardiopulmonary coupling under parasympathetic control. When the heart rate increases the RSA will decrease, and vice versa (Moldovan et al. 2004). An increase of RSA (independent of the respiratory frequency) with a decrease in heart rate can be interpreted as an effect of increased cardiac vagal tone (Barlow, 2007). RSA tells essentially nothing about the general central vagal road. It is important that the heart rate is at least twice as fast as the ventilation (Nyquist frequency). In case the ventilation is greater than the half of the heart rate RSA has no longer any meaning (Aliasing frequency) (Grossman and Taylor, 2007).

RSA can be extracted in various ways from an ECG: ECG-derived heart period (IBI), through the heart rate, the peak-valley method (straightforward method) (Schmidt and Segalowitz, 2007). From the ECG derived respiration there (EDR) four different parameters can be obtained (ECGfilt, Rampl, RSampl, AQRS). Widjaja et al. (2010) concluded that Rampl would be the best approach for the respiratory signal. Through the peak-valley method the RSA is extracted by means of subtracting of the shortest IBI (associated with respiratory inspiration) in ratio with the longest IBI (associated with respiratory expiration) (Cacioppo et al. 2007; Grossman et al. 1990), provided that the shortest IBI (highest heart rate) is a component of the acceleration range of the longest IBI (lowest heart rate) of the deceleration series (van Dijk et al. 2013). The 'golden standard' for measuring RSA continues to be the direct measurement of the aire flow through the nose and mouth (Lamberti and de Bie, 2003).

Both HRV software programs quantify RSA from the ECG signal. VU-DAMS v3.9 uses the peak-valley method (ms2), while Kubios v2.2 uses EDR (Hz).

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8 Part V: methodology and measurements The research method for the RILHOU-RCT is described in detail in the â&#x20AC;&#x2DC;Common documentsâ&#x20AC;&#x2122; (De Cock, 2014).

8.1 Measurements The length of every subject was determined by using a length meter (Dencor ADE MZ10017 tape measure, 0-2200 mm) that was attached to the wall. The belly circumference was measured with a Dencor ADE MZ10021 Circumference and Length Measuring Band (50-1500 mm). BMI was calculated by using a Tanita Bioelectrical Impedance Analysis scales. The validity and reliability of this device is already described elsewhere (Wang and Hui, 2015).

Relative air humidity level and the difference between the inside and outside temperature are potential confounders for heart rate and skin conductance (Posthumus et al. 2009) and HRV (De Cock, 2014). The ambient temperature and relative humidity level was measured by a weather station. Data from the outdoor temperature were not acquired (De Cock, 2014).

ECG, ICG and SCL was obtained with the VU-AMS 5fs device. The validity and reliability of the VU-AMS device is already described elsewhere (de Geus et al. 1995; Willemsen et al. 1996). HRV parameters were obtained from the ECG in accordance with the guidelines of the Task Force of the European Society of Cardiology and North America Society of Pacing Electrophysiology (Task Force, 1996a,b, Sassi et al. 2015), as well as RSA and respiration rate. Pre-ejection period (PEP) was derived from the ICG. Skin conductance level was obtained from the electrodermal activity. Blood pressure was obtained with a digital blood pressure monitor (Omron M6 HEM-7001-E; Arm cuff: Medium, arm circumference 22-32 cm) (De Cock, 2014). The validity and reliability of this device has already been described elsewhere (Altunkan et al. 2008, Altunkan et al. 2007).

8.1.1 Research This study describes the ethical and legal guidelines based on the Belgian law of 7 May 2004 concerning experiments on the human person (descriptive research, part I). A

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descriptive comparison of the HRV parameters between software program VU-DAMS (v3.9) and Kubios (v 2.2) has been elaborated (part IV).

In addition, the effect of a set of indirect osteopathic techniques on HRV, SCL, RSA, PEP and blood pressure in healthy Dutch adults between 20 and 50 years is being investigated (efficiency study). Blood pressure serves only as clinical effect size while HRV, SCL, RSA, respiration rate and PEP act as an intermediate effect size. The primary purpose of this study is limited to the formulation of statements regarding the efficiency of the intervention (De Cock, 2014).

8.1.2 Research title Effects of a set of cranial techniques on the autonomic nervous system in a cohort of healthy adults.

8.1.3 Research question Have indirect osteopathic cranial techniques a direct or short-term (treatment) effect on healthy Dutch-speaking adults on: HRV, SCL, RSA, PEP and blood pressure?

8.2 Hypotheses The aim of the null hypothesis significance testing (H0) is to establish specific the likelihood of an effect and/or establish a pattern of order (both requires ruling out equivalence). It is not the goal to establish some quantitative values, because then is the H0 methode not the best choice (Pernet, 2015). The hypothesis cited specifically refers to "The applied techniques". The author refers to a set of indirect osteopathic cranial techniques (Infra: 8.2.1).

8.2.1 Hypothesis related to HRV H0: The applied techniques have no immediate or short-term (treatment) effect on HRV. H1: The applied techniques have a immediate or short-term (treatment) effect on HRV.

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8.2.2 Hypothesis relating average SCL H0: The applied techniques have no immediate or short-term (treatment) effect on average SCL.

H1: The applied techniques have a immediate or short-term (treatment) effect on average SCL.

8.2.3 Hypothesis related to RSA H0: The applied techniques have no immediate or short-term (treatment) effect on RSA. H1: The applied techniques have a immediate or short-term (treatment) effect on RSA.

8.2.4 Hypothesis related to PEP H0: The applied techniques have no immediate or short-term (treatment) effect on PEP. H1: The applied techniques have a immediate or short-term (treatment) effect on PEP.

8.2.5 Hypothesis related to blood pressure H0: The applied techniques have no immediate or short-term (treatment) effect on blood pressure.

H1: The applied techniques have a immediate or short-term (treatment) effect on blood pressure.

8.3 Material and methods The VU-AMS device was described in detail (part III). The other measuring instruments were already mentioned. All the measurements (length, BMI, blood pressure, ECG/ICG, EDA) were performed according to standardized protocols. These protocols can be consulted in the â&#x20AC;&#x2DC;Common documentsâ&#x20AC;&#x2122; (De Cock, 2014) and RILHOU-RCT DRAAIBOEK + FMEA (werkdocument).

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8.3.1 Physiological recording from the VU-AMS 5fs device ICG/ECG electrodes (Kendall ARBO H98SG) were placed carefully in order to obtain an optimal lead II distraction. This results in a prominent R-wave peak and a clear Twave amplitude. The skin was disinfected with alcohol disinfectant or hibidil (for allergic reaction with alcohol disinfectant) before the electrodes were placed, resulting in a low electrode resistance.

The first ECG electrode (V-) was placed below the right clavicle, four centimeters lateral to the sternum. The second ECG electrode (V+) was placed between the right costae eleven and twelve. The third ECG electrode (V+) was placed on the left lateral side of ninth costea (apex of the heart), at the level of the processus xiphoideus.

The first ICG electrode (V-) was placed on the top of the incisura jugularis flexura sternalis. The second ICG electrode (V+) was placed on the processus xiphoideus.

Two generating electrodes were placed on the spine: one electrode (I-) on cervical vertebral C4, at least three centimeters above the first ICG electrode (V-). A second electrode (I+) was placed on the thoracic vertebra (T8-T9), at least three centimeters below the second ICG electrode (V+) (van Dijk et al. 2013).

The ECG signal was measured and transformed with a sampling frequency of 1000 Hz (16-bit resolution). For the ICG, two generating electrodes (I- and I+) supplied a constant electric current of 350 ÂľA (50 KHz) through the subject (Cybulski, 2011). The power was sampled at a frequency of 1000 Hz for dZ, and 250 Hz for Z0.

EDA was obtained by an exosomatic measurement, with direct current. SCL was measured with the method of 0.5 V constant voltage (Fowles et al. 1981; Roth et al.

2012; Benedek and Kaernbach, 2010) with a range between 0-95 ÂľS. The sample rate for skin conductance measurement was fixed at 10 samples per second (10 Hz). The VU-AMS 5fs device produced a digital conversion for measure skin conductance by using a 16 bit AD-converter. On the two skin conduction Ag-AgCl electrodes (65 mm diameter) Biopac isotonic 4OZ gel was spreaded, precisely until the SC electrodes were covered. The Ag-AgCl electrodes were placed at the volar side of the dominant hand on the level of the intermedial falanx (index and digitus medius) (Dawson et al.

2007).

The VU-AMS 5fs device was placed on the treatment table next to the left side of the subject.

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8.3.2 Data reduction ECG, ICG and EDA data was imported into the software program VU-DAMS v3.9. The interpolation of the R-wave in the ECG was sampled at a frequency of 1000 Hz. A cubic spline function was applied with a sampling frequency of 4 Hz. Standard sigma (σ) 3.5 was used to remove artifacts in the settings of the QRS algorithm.

Software program AmsQRS did perform automatically three analyses (artefact detector; R-peak detector; suspicious beats detector) on the ECG fragments. A visual inspection for possible ectopic beats of the R-wave peaks and the IBI (ms) was conducted through AmsGraph. Missed or incorrect R-wave peaks (short/long beats) were manually examined and, if necessary, corrected. Software program AMSASC was used to convert the binary VU-DAMS data to an ASCII format. VU-DAMS does not provide nonlinear HRV analysis, therefore the software program Kubios v2.2 was applied. Artifacts were removed for the data analysis.

An automatic and manual SCL artifact detection was performed. A low pass filter with a cut-off frequency of 2 Hz was applied to filter noise and power line interference of the SCL signal. The filtering took place in forward and reverse direction, this to avoid the shifting of peaks. Artifacts were removed before the data analysis.

Software AMSIMP was used to analyze the ICG recording. Automatically an algorithmic score was performed before the detection of the upstroke (B-point), dZ/dt min (C-point) and incisura (X-point). The cardiac pre-ejection period was marked in the ICG between the Q-golf onset and B-point. Corrections were carried out manually where necessary. All the data were manually inspected by one researcher (Farah et al.

2016). A random sample on 10 datasets was externally examined to detect and control the PEP points and the ECG artifacts.

The raw ECG data (ASCII format) was uploaded in Kubios v2.2 for further HRV analysis. A cubic spline function was performed with a sample rate of 4 Hz. An automatic (low) artifact detection was performed on the QRS algorithm.

Both software programs use the Welch's periodogram method (FFT) for HRV analysis in the frequency domain, with a standard signal preprocessing setting of the R-R interval detrending ‘smoothness priors matrix' Lambda 500 (fc= 0.035 Hz). Frequency bands LF (0.04-.15 Hz) and HF (0.15-.4 Hz) were used as recommended by the Task Force (1996). The option "spectral factorization” was not used in Kubios v2.2.

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8.3.3 Study type Study type:

Non-commercial, monocentrisch Interventioneel experiment

Study design:

Allocation: Randomized Clinical Trial

Endpoint classification:

Efficiency study

Intervention Model:

Parallel assignment

Masking:

Double blind (subject, investigator, statistician)

Primary purpose RILHOU-RCT:

Effectiveness study

8.3.4 Study progress A request for advice for the RILHOU-RCT to assess the ethical aspects and privacy of the subjects was submitted to the CME ZOL (fully recognized). After a powerpoint presentation of the RILHOU-RCT and answering the reply “questions and comments” by the CME ZOL the final approval was granted for the study (Eudract/B-nr.

B371201422023). The legal mandatory concerning 'civil liability' insurance for experiments on the human person was granted by Ethias (Policy number: 45.326.306).

Specific for this study, an additional clause "Proefschrift Osteopathie aan FICO” was included in the civil liability insurance policy for Wens Chris granted by insurance company ‘AMMA’. The study protocol together with the request for advice CME, the temporary negative advice CME, reply on the ‘questions and comments’ of the CME, the finally positive advice CME, the document with regard to Ethias insurance and further correspondence was merged in the form of 'Common documents'. Before the start of this study, a risk and safety analysis (FMEA) was conceived by Wens C (document: RILHOU-RCT DRAAIBOEK + FMEA [werkdocument]) in collaboration with an external safety advisor (Janssens P) and was subject in a preliminary study.

The subjects were recruited to participate in this study (13 June 2015 to 5 July 2015) by flyers and social media. Subjects were selected on the basis of inclusion and exclusion criteria by means of a (randomized) questionnaire. The subjects were divided per software-generated randomization into an experimental group (R) or Sham group

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(S) and paired with a code prior to R or S (Addendum 3). This code was included into the investigation file.

The subjects were expected 30 minutes before the start of the study in the waiting room of the research center (FICO). The 'ICF' and 'letter of agreement and consent form' was signed before the start of the study by the subject and investigator. In the agreement the subject explicitly wrote "read and approved" by the subject.

The questionnaire was reviewed together with the subject and an additional question was added related to pain. This question was scored on a categorical base line scale (0-10). Length, BMI and belly circumference was measured (one-time) before the beginning of the experiment. The ambient temperature and relative humidity level was measured at the beginning of the experiment and at the start of the follow-up. Blood pressure was measured (left and right) before and after the experiment, resp. followup. ECG, ICG and EDA were measured with the VU-AMS 5fs device during the experiment/follow-up. The ECG recording was based on a bipolar extremity distraction.

The ICG recording was obtained by using a tetrapolaire method. EDA was obtained on the basis of an exosomatic measurement. All the measurements were described in detail in standard protocols (Common documents and RILHOU-RCT DRAAIBOEK + FMEA [werkdocument]) (De Cock, 2014).

During the experiment/follow-up the subject was in a supine position on the treatment table, except for the intermediate period (subject walked around in the room of the investigation). The experiment contains in total 5 time periods (Table 6).

Table 6: Time scale experiment Rest period (label 1)

Pre measurement (label 2)

Intermediate period (label 3)

5 min

10 min

5 min

Post Experiment/Sham measurement (label 4) (label 5) 10 -15 min

10 min

In the experimental group six successive indirect osteopathic cranial techniques, were performed. In the Sham group were three consecutive hands-on techniques used. All the techniques were carried out according to a standard protocol. The follow-up (Table 7) took place 6-10 days after the experiment (De Cock, 2014).

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Table 7: Time scale follow-up Rest period (label 1)

Control measurement (label 2)

5 min

10 min

Possible side effects (adverse events) during the experiment and follow-up were observed and noted in the investigation file.

8.3.5 Population The research population in this study consisted of 40 healthy dutch speaking mental competen adults between 20 and 50 years old (Addendum 3 and 8).

8.3.5.1Inclusion criteria Dutch speaking mentally competent adults between 20-50 years, BMI ≤ 30, in good health, the ability to lie down (pain free) in a supine position with both legs straight. At least one hour before the experiment haven’t smoked (Minami et al. 1999) or drunk coffee (Rauh et al. 2006; De Cock, 2014; De Cock, 2015).

8.3.5.2Exclusion criteria Non-native adults, not mentally competent, < 20 years or > 50 years (Voss et al. 2015), BMI > 30 (Farah et al. 2013), > 15 glasses of alcohol per week (Ingjaldsson et al.

2003), smoking: > 15 cigarettes per day (Alyan et al. 2008; Barutcu et al. 2005), use of drugs (medication): antidepressants (Penninx et al. 2008; Noordam, 2015; Licht and Penninx, 2009), cardiac problems (Kors et al. 2007; Hillebrand et al. 2013), diabetes (Kudat et al. 2006; Schroeder et al. 2005), pregnancy (Van Leeuwen et al. 1999; Brändle et al. 2015; Chamchad et al. 2007); Familial hypercholesterolemia (Sung, 2010), epilepsy (Lotufo et al. 2010), neurological disorders, students FICO (De Cock, 2014).

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8.3.6 Investigation This study investigates the effect of a set of indirect osteopathic cranial techniques on 'HRV, SCL, RSA, PEP and blood pressure' in healthy Dutch-speaking mentally competent adults between 20 and 50 years with a BMI â&#x2030;¤ 30.

8.3.7 Procedure The modus operandi of the experiment was described in the Common documents and RILHOU-RCT DRAAIBOEK + FMEA (werkdocument). Both documents were legally registered in the Royal Library in Brussels (De Cock, 2014). All subjects permitted through the document 'Letter of agreement and consent form' their written consent before the start of the experiment.

8.3.7.1Randomization The questionnaires were randomized (9 sets of 40 unique order of the questions numbers. Set 10 is the original [non-randomized] questionnaire). An independent investigator (scientific coordinator) makes a random allocation by using computergenerated random numbers (v.4/dd 26/01/2015/ user JDC; 40 unique numbers per set). The sample was divided with blind allocation into 2 groups: experimental group (R) and Sham group (S) (De Cock, 2014; Jeehyoung and Wonshik, 2014).

8.3.7.2Sham Three hands-on techniques were used in following order (at the level of: the head, the umbilicus and feet). The subject was during the Sham in a supine position on the treatment table. The researcher focused during the performance on the contact places (De Cock, 2014).

8.3.7.2.1

Technique at the height of the head

The researcher took for 4 minutes place at the head of the subject and placed his both thumbs at the sutura sagittalis. Both palms were in contact with the ossis parietalis.

The other four fingers were pointed towards the face.

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8.3.7.2.2

Technique at the level of the umbilicus

The researcher took place for 4 minutes on the right side of the subject, with his face turned towards the subject. The right hand was placed at the level of the umbilicus, with the palm of the hand in the middle of the abdomen. The fingers were directed cranially.

8.3.7.2.3

Technique at the height of the feet

The researcher took place for 4 minutes at the height of the feet of the subject. Both thumbs were placed just below the facies articularis malleoli medialis tibiae. Both index fingers were placed on the inferior side of the facies articularis malleoli lateralis fibulae.

Both palms of the hands and fingers were placed on the forefoot along the lateral side.

8.3.7.3 Experimental techniques The subjects lay in a supine position on the treatment table, with their head in neutral position. A detailed description of the applied techniques can be found in handbooks of osteopathy. The applied techniques were also described in detail in ‘Common documents’ under ‘Gebruikte technieken in the RILHOU-RCT' (De Cock, 2014; Addendum 10).

8.3.7.3.1

Techniques experiment according to sequence of

execution -

Third Vault Hold (Upledger and Vredevoogd, 1983)

-

Spheno-basilar decompression (Liem, 2004; Attlee, 2012; Upledger and Vredevoogd, 1983)

-

J. E. Upledger’s ‘ear pull’ technique (Liem, 2004)

-

Parietal lift (Liem, 2004; Chiatow, 1999; Landouzy, 1993; Upledger and Vredevoogd, 1983; Magoun, 1976)

-

Frontal lift (Liem, 2004; Chiatow, 1999; Landouzy, 1993, Upledger and Vredevoogd, 1983)

-

Technique for the frontonasal suture (Liem, 2004; Chiatow, 1999)

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8.3.7.4Blinding The subject and the performing researcher were blinded before the randomization concerning the subjectâ&#x20AC;&#x2122;s allocation into the groups (R/S). The performing researcher was blinded for the implementation of the experimental/Sham techniques just before he was present with the subject. By means of a signal (paper with the letter R of S on the table) the researcher knew which techniques he had to use for the specific subject. The performing researcher was blinded for the data collection during the total period of the investigation. The performing researcher and the subjects were being coded whereby the blinding remained guaranteed for the statistician and other persons (De Cock, 2014).

8.3.7.5Processing 8.3.7.5.1

Description of the variables

Fifty-two variables were analyzed per group (R/S) in SPSS performed by an external statistician. The variables were divided for both groups into eight categories (subject variables, HRV, SCL, RSA, respiration rate, PEP, blood pressure, ambient temperature). Subject variables (gender, age, height, weight, BMI, body fat percentage, belly circumference) were analyzed one time only. HRV was subdivided according to the analysis domain (time domain, frequency domain, nonlinear analysis). Used abbreviations with elucidations are presented in Table 8.

HRV, SCL, RSA, respiration rate and PEP were analyzed during the different time periods (post measurement experiment/Sham (t1), follow-up experiment/Sham (t2)) to compare with the pre measurement experiment/Sham (t0). Both groups served as their own control (t0) (Table 6 and 7).

Blood pressure was divided into systolic blood pressure (SBP) and diastolic blood pressure (DBP), likewise left (L) and right (R). The analysis was performed over four time periods: pre measurement experiment/Sham (control) (t0), post measurement experiment/Sham (t1), pre measurement follow-up experiment/Sham (t3), post measurement follow-up experiment/Sham (t2). Ambient temperature (KT) was analyzed by comparing the follow-up experiment/Sham (KT follow-up t2) with the experiment/Sham (RT Test t1).

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8.3.7.5.2

Descriptive statistics

In order to have an insight into the distribution of the variables of both groups, a quantitative description of the average characteristics was performed per variable (frequency domain distribution, central tendency 'mean', dispersion 'standard deviation' and variance). For some variables extra analyses were performed for the range (minimum–maximum), skewness and kurtosis (by using Microsoft Office Excel 2007).

The standard deviation gives additional information/ details on the average on ‘how far the values are spread from each other’. The larger the standard deviation, the more "space" there is present between the numbers.

The coefficient of variation of the observations was used to describe the level of variability within-subjects (intra-individual variability) and between subjects (interindividual variability). 95% confidence interval was calculated because they give more information than p-values (Rümke, 1989). In contrast to p-values, confidence intervals indicate the direction of the effect studied. Conclusions about statistical significance are possible with the help of the confidence interval (du Prel et al. 2009).

Only variables with a (treatment) effect will be extra examined to avoid 'regression to the mean' (Morton and Torerson, 2003). 'Regression to the mean' is a general statistical phenomenon that can lead to serious implications for erroneous conclusions, if an effect is due to a treatment or if it’s due to a chance (Yu and Chen, 2015; Morton and Torerson, 2003).

Skewness is the measure that indicates if a skewness is divided into left- or right, compared with the normal (or Gaussian) distribution. For distributions that are normally distributed (symmetric distribution), the skewness parameter is zero (No skew). This means that the distribution is perfectly normal divided, which is not the case in this study. A positive skew is characterized by observations on the right side and are further away from the center than on the left side (right skewed), opposite for a negative skew (left skewed). Kurtosis characterizes the measure of the ‘tailedness’ of the probability distribution of a real-valued random variable. In case of a normal distribution the value of kurtosis is 0 (mesokurtic). A negative outcome (-) indicates a flatter distribution than the normal distribution (platykurtic). A positive outcome (+) indicates a sharper more peaked (tailedness) distribution (leptokurtic) than the normal distribution (mesokurtic).

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8.3.7.5.3

Normality test

A normal distribution of the variables was applied for the possible performance of parametric tests. Among the non-normal distributed variables non-parametric tests (distribution-free tests) were applied.

The Kolmogorov-Smirnov Test (applied with Lilliefors significant correction) and the Shapiro-Wilk Test were both performed to investigate the significant differences between the observations of the different time periods and groups in the preliminary stage (baseline). The Shapiro-Wilk Test was used for small sample sizes (< 50 samples) and large sample sizes (2000 samples). Given the amount of variables this test was used to determine the normality of the variables. The Shapiro-Wilk test is more powerful as a normality test, compared with the Kolmogorov-Smirnov Test (with Lilliefors significant correction) (Razali and Wah, 2011).

The subject variables were all normally distributed (p >.05), but an important part of the other variables exhibited some significance (p >.05). Parametric tests (Independent Ttest and Pearson Chi-square test) were performed for the subject variables (gender, age, length, weight, BMI, body fat percentage, belly circumference). On the other variables (HRV, SCL, RSA, respiration rate, PEP, blood pressure, ambient temperature) nonparametric tests (Wilcoxon Signed Ranks Test and ANCOVA) were performed.

8.3.7.5.4

The Independent T-test (Student's T-test)

The Independent T-test was used to test the differences between the two groups (R/S).

The subject variables were scaled as ratio and considered as independent because the two different populations were compared at the start of the study (Baseline).

8.3.7.5.5

The Pearson Chi-square test (point-of-care test)

The Pearson Chi-square test was performed because â&#x20AC;&#x2DC;genderâ&#x20AC;&#x2122; consists of two categorical variables (male or female).

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8.3.7.5.6

Differences between the same group (treatment

variables) on different time periods (Wilcoxon Signed Ranks Test) The Wilcoxon Signed Ranks Test (paired-samples t-test) determines whether if one of the two partitions have systematically larger (or smaller) values than the other partition.

It is an equivalent of the Dependent T-test and was applied for the other variables. The Wilcoxon Signed Ranks Test compares the scores of the variables that come from the same subjects (dependent results). This test examines whether or not changes did occur in the scores of the variables from one period to another, while maintaining the same scale.

A significant effect was found for variables in the experimental group when the resulting p-value was <.05, compared with a p-value >.05 in the Sham group (Dahiru, 2008). A ‘trend or tendency’ was found in the experimental group for the variables when the pvalue was between >.05 and <.15, compared with a p-value >.05 in the Sham group (arbitrary choice). For a Sham effect or possible tendencies the opposite supra was handled (surpa experimental group). There isn’t any unequivocally in the existing literature about the terminology of ‘trend or tendency’ (Gibbs and Gibbs, 2015), perhaps the demarcation of the p-values are also controversial (Knuffner and Walker, 2016).

Z-values (SD-unit) are based on positive ranks (the average values are decreased, compared with the pre-measurement) and negative ranks (the average values are increased, compared with the pre-measurement).

The 'Wilcoxon Signed Ranks Test' on its own is not completely accurate enough to formulate statements about a significant (treatment) effect. An ANCOVA (ANOVA with covariaat) was performed to investigate if the relationship between the qualitative variable X and the quantitative variable Y wasn’t influenced by a quantitative variable Z (t0 measurement).

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8.3.7.5.7

The difference between the different groups on

different time periods (ANCOVA) An ANCOVA test (analysis of covariance) was performed to determine if there were significant differences between the major average of two or more independent (nonrelated/independent) groups (in particular for the custom average). An ANCOVA allows a "statistical control" of a third (disturbing) variable (covariate), which possibly may adversely affect the results. The t0 measurement (control group experiment/Sham) was taken as "covariate variable" and compared through the groups. In this way the difference that might exist between the groups (for the tested variable) was eliminated.

The partial eta squared (ηp2) was calculated to express the effect size on the strength of the found difference (small effect size when ηp2= 0.01; a medium effect size when ηp2= 0.06; a large effect size when ηp2= 0.14) (Cohen, 1988).

An ANCOVA analysis is a linear model, which implies that this analysis was applied for each individual variable.

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9 Part VI: Results Table 8: Follow abbreviations were used for the different variables Blood pressure t0 t1 t3 t2 SBPL SBPR DBPL SBPR

controle group (pre measurement) post measurement pre measurement follow-up post measurement follow-up systolic blood pressure left systolic blood pressure right diastolic blood pressure left diastolic blood pressure right

HRV analysis [V] [K] t0 t1 t2 (ms) (bpm)

VU-DAMS Kubios controle group post measurement follow-up milliseconds beats per minute

Time domain HR IBI SDNN rMSSD pNN50

heart rate interbeat interval standard deviation of normal to normal intervals root mean square of successive differences the proportion of NN50 divided by total number of NNs.

Frequency domain LF low frequency HF high frequency [FFT] fast fourier transform [AR] autoregression n. U. normal units 2 ms square millisecond (Hz)Hertz Non-lineaire analysis DFA detrended fluctuation analysis ApEn approximate entropy SD1

dispersion (standard deviation) of points perpendicular to the axis of line-ofidentity

SD2

dispersion (standard deviation) of points along the axis of line-of-identity

ShanEntropy

shannon entropy

D2

correlation dimension

Other RSA

respiratory sinus arrhythmia

PEP SCL

pre ejection period skin conduction level

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9.1 Parametric test for the subject variables (Independent Ttest and Pearson Chi-square test).

9.1.1 The difference between the experimental group (R) and the Sham group (S) for the subject variables, scaled ratio (Independent T-test).

Table 9: Independent T-test Independent Samples Test Levene's Test for Equality of Variances F Age

Equal variances assumed

,613

Sig. ,439

Equal variances not assumed Lenght[cm)]

Equal variances assumed Equal variances assumed Equal variances assumed Equal variances not assumed

Body Fat%

Equal variances assumed

,465

,499

Equal variances not assumed PerimeterBelly[cm] Equal variances assumed

,810

,500

2,071

-3,692

,810

,500

2,071

-3,692

4,692

38

,148

-3,79500

2,57038

-8,99846

1,40846

-1,476 37,953 ,965 ,840

,001

4,692

,148

-3,79500

2,57038

-8,99867

1,40867

38

,385

-3,22500

3,66623 -10,64690

4,19690

-,880 37,977

,385

-3,22500

3,66623 -10,64705

4,19705

,078

38

,939

,07000

,90154

-1,75506

1,89506

,078 37,998

,939

,07000

,90154

-1,75507

1,89507

-,408

38

,686

-1,03500

2,53626

-6,16940

4,09940

-,408 37,899

,686

-1,03500

2,53626

-6,16985

4,09985

38

,176

-4,09500

2,96884 -10,10510

1,91510

-1,379 37,787

,176

-4,09500

2,96884 -10,10621

1,91621

-,880

,977 -1,379

Equal variances not assumed

df

95% Confidence Interval of the Lower Upper

38

,002 ,041

Mean Std. Error Sig. (2-tailed) Difference Difference

,241 37,960 ,817 -1,476

Equal variances not assumed BMI

t ,241

,054

Equal variances not assumed Weight[kg]

t-test for Equality of Means

There was no significant difference found in the 'Baseline' between the two groups for the different subject variables (Table 9). The two groups are permitted be compared.

9.1.2 The difference between the experimental group and the Sham group for the subject variables, scaled nominal (The Pearson Chi-square test).

Only gender was nominally scaled. There was no significant difference found for gender in the 'Baseline' between the two groups (Table 10).

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Table 10: Pearson Chi-Square Test

9.2 Non-parametric test for the other variables (Wilcoxon Signed Ranks Test and ANCOVA).

9.2.1 Summary results Wilcoxon Ranks Test and ANCOVA 9.2.1.1Comparison baseline (t0) experimental group with Sham 9.2.1.1.1

Total variance of HRV

The experimental group exhibited a higher general Total variance of HRV (Total power ms2: FFT: 14.99%, 3090.69 ± 3974.73, 95% CI [1348.69 – 4832.69], AR: 16.17%, 3250.60 ± 4206.92, 95% CI [1406.84 – 5094.36]; SDNN ms: 4.93%, 66.78 ± 34.39, 95% CI [51.70 -81.85]), compared with the Sham (Total power ms2: FFT: 2627.36 ± 3236.51, 95% CI [1208.90 – 4045.83], AR: 2724.97 ± 3137.76, 95% CI [1349.79 4100.16]; SDNN: 63.49 ± 27.20, 95% CI [51.56 – 75.41]. The experimental group showed a greater variety for Total power ms2 (FFT: CVg: 1.29%, AR: CVg: 1.29%) and SDNN ms (CVg: 0.51%) between the subjects, compared with the Sham (Total power ms2: FFT: CVg: 1.23%, AR: CVg: 1.15%; SDNN ms: CVg: 0.43%). The experimental group showed a lower degree in ApEn and a increase of D2 (See infra: 9.2.1.6: Cardiac complexity and regularity).

9.2.1.1.2

Parasympathetic activity

In the experimental group, the ANS was general characterized with an increased modulation of parasympathetic activity (rMSSD ms: 15.66%, 66.93 ± 65.93, 95% CI [38.04 – 95.83]; pNN50 %: 9.77%, 29.62 ± 22.41, 95% CI [19.80 – 39.80]), compared with the Sham (rMSSD ms: 56.45 ± 37.53, 95% CI [40.00 – 72.90]; pNN50 %: 26.72 ± 22.65, 95% CI [16.80 – 36.65]). The variation between the subjects was greater in the

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experimental group for rMSSD ms (Expi.: CVg: 0.99) and small for pNN50 % (CVg: 0.76), compared with the Sham (rMSSD ms: CVg: 0.66; pNN50 %: CVg: 0.85).

An increased modulation of parasympathetic activity was confirmed in the experimental group by the increase of the rapid beat-to-beat RR-interval (short-term) variability (SD1 ms: 9.45%, 42.23 ± 34.02, 95% CI [27.32 -57.14]), compared with the Sham (SD1 ms: 38.24 ± 24.63, 95% CI [27.44 – 49.04]. The experimental group showed a greater variation between the subjects for SD1 ms (Expi.: CVg: 0.81), compared with the Sham (CVg: 0.64).

In the experimental group, an increase of the cardiac vagal control (RSA: 8.57%, 82.22 ± 43.67, 95% CI [63.08 – 101.37]; HF ms2: FFT: 7.47%, 1296,09 ± 1343.30, 95% CI [707.37 – 1884.82], AR: 28.80%, 1712.97 ± 2645.50, 95% [553.53 – 2872.41]; HF n.

U.: FFT: 7.77%, 49.86 ± 14.98, 95% CL [43.29 – 56.42], AR: 8.55%, 49.75 ± 17.58, 95% CI [42.05 – 57.46]) was accompanied and found by a reduced respiratory frequency (-1.19%, 15.81 ± 2.85, 95% CI [14.56 – 17.06]) (ECG derived), compared with the Sham (RSA: 75.18 ± 33.55, 95% IC [60.48 – 89.88]; HF ms2: FFT: 1199.30 ±1493.42, 95% CI [544.78 – 1853.82], AR: 1219.70 ± 1423.10, 95% CI [596.00 – 1843.40]; HF n. U.: FFT: 45.98 ± 15.81, 95% CI [39.06 – 52.91], AR: 45.50 ± 14.38, 95% CI [39.19 – 51.80]; respiration rate bpm: 16.00 ± 2.70, 95% CI [14.82 – 17.18]).

The experimental group showed a larger variation between the subjects for RSA (CVg: 0.53), HF ms2 AR (CVg: 1.54), HF n. U AR (CVg: 0.35) and respiration rate bmp (CVg: 0.18) while the FFT analysis showed a small variation (HF ms2: CVg: 1.04, HF n. U.: CVg: 0.30), compared with the Sham (RSA: CVg: 0.45, HF ms2: FFT: CVg: 1.25, AR: CVg: 1.17, HF n. U.: FFT: CVg: 0.34, AR: CVg: 0.32, respiration rate bpm: CVg: 0.17).

9.2.1.1.3

Sympathetic activity

The experimental group exhibited a higher sympathetic cardiac control (PEP ms: 0.10%, 90.64 ± 19.71, 95% CI [82.00 – 99.27] with a heightened state on the physiological arousal (SCL µS: 23.41%, 4.26 ± 3.42, 95% CI [2.76 – 5.76], compared with the Sham (PEP ms: 90.55 ± 16.24, 95% CI [83.43 – 97.67]; SCL µS: 3.26 ± 1.29, 95% CI [2.70 – 3.83]).

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The experimental group showed a larger variation between the subjects for PEP ms (CVg: 0.22) and SCL µS (CVg: 0.80), compared with the Sham (PEP ms: CVg: 0.18, SCL µS: CVg:0.40).

9.2.1.1.4

Sympathovagal balance

The experimental group showed a reduced activity on the baroreceptors and between the mix of the ANS (LF ms2: FFT: -26.67%, 1155.84 ± 1225.39, 95% CI [618.79 – 1692.89], AR: -3.05%, 1322.77 ± 1464.94, 95% CI [680.73 – 1964.81]; LF n. U.: FFT: 7.69%, 50.06 ± 15.02, 95% CI [43.47 – 56.64], AR: -3.67, 50.17 ± 17.62, 95% CI [42.45 -57.89]) and on the autonomic cardiovascular regulation system (LF/HF ratio ms2: FFT: -21.88%, 1.26 ± 0.96, 95% CI [0.84 – 1.68], AR: -9.59%, 1.36 ± 1.12, 95% CI [0.87 – 1.84], compared with the Sham (LF ms2: FFT: 1464.06 ± 2093.01, 95% CI [546.76 – 2381.36], AR: 1363.17 ± 1766.14, 95% CI [589.12 – 2137.21]; LF n. U.: FFT: 53.91 ± 15.84, 95% CI [46.96 – 60.85], AR: 52. 01 ± 18.84, 95% CI [43.75 – 60.27]; LF/HF ratio ms2: FFT: 1.53 ± 1.23, 95% CI [0.99 – 2.07], AR: 1.49 ± 1.05, 95% CI [1.03 – 1.94].

The experimental group showed a small variation between the subjects for LF ms2 (FFT: CVg: 1.06, AR: CVg: 1.11), LF n.u. AR (CVg: 0.35), LF/HFratio ms2 FFT (CVg: 0.76) while a large variation was seen for LF n.u.FFT (CVg: 0.30) and LF/HF ratio ms2 AR (CVg: 0.82), compared with the Sham (LF ms2: FFT: CVg: 1.43, AR: CVg: 1.30; LF n. U.: FFT: CVg: 0.29, AR: CVg: 0.36; LF/HF ratio ms2 : FFT: CVg: 0.80, AR: CVg: 0.70).

9.2.1.1.5

Scaling behaviour of the ANS

A reduction was found in the experimental group for the short-term fluctuation (DFA α1: -1.62%, 0.95 ± 0.26, 95% CI [0.84 – 1.07) and long-term fluctuation (DFA α2: -4.52%, 0.25 ± 0.09, 95% CI [0.21 -0.29]) with an increasement of the short-term variability (SD2 ms: 4.40%, 60.15 ± 32.39]), compared with the Sham (DFA: α1: 0.97 ± 0.24, 95% CI [0.86 – 1.07], α2: 0.26 ± 0.11, 95% CI [0.21 – 0.30]; SD2 ms: 57.50 ± 25.72, 95% CI [46.23 – 68.78].

The experimental group showed a larger variation between the subjects for DFA α1 (CVg: 0.28) and SD2 (CVg: 0.54) while a small variation was seen for DFA α2 (CVg: 0.37), compared with the Sham: (DFA: α1: CVg: 0.24, α2: CVg: 0.42; SD2: 0.45).

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9.2.1.1.6

Cardiac complexity and regularity

The experimental group showed a lower degree in the complexity of the regularity of the physiological time series (ApEn: -2.18%, 1.28 ± 0.12, 95% CI [1.23 – 1.33] with a higher degree in the global autonomous variability (D2: 12.32%, 2.94 ± 1.72, 95% CI [2.18 – 3.69]), compared with the Sham ( ApEn: 1.31 ± 0.12, 95% CI [1.25 – 1.36]; D2: 2.58 ± 1.52, 95% CI [1.91 – 3.24]). Both groups showed the same variation for ApEn between the subjects (CVg: 0.09). The experimental group presented a greater distribution of the physiological patterns (ShanEntropy: 1.59%, 3.07 ± 0.31, 95% CI [2.93 – 3.20]), compared with the Sham (3.02 ± 0.24, 95% CI [2.91 – 3.13]).

There was a small variation between the subjects in the experimental group for D2 (CVg: 0.58) and a large variation for ShanEntropy (CVg: 0.10), compared with the Sham (D2: CVg: 0.59; ShanEntropy: CVg: 0.08).

9.2.1.1.7

Blood pressure

The systolic and diastolic blood pressure was lower in the experimental group (SBPR mm Hg: -3.38%, 117.00 ± 11.68, 95% CI: [111.88 – 122.12]; DBPR mm Hg: -6.82%, 74.05 ± 7.63, 95% CI [70.71 – 77.39], compared with the Sham (SBPR mm Hg: 120.95 ± 17.47, 95% CI [113.29 – 128.61], DBPR mm Hg: 79.10 ± 10.59, 95% CI [74.46 – 83.74].

The experimental group showed a small variation between the subjects for SBPR mm Hg (CVg: 0.10) and DBPR mm Hg (CVg: 0.10), compared with the Sham (SBPR mm Hg: CVg: 0.14; DBPR: 0.13).

9.2.1.2 t1 and t2 compared with the baseline (t0) in the same group (Wilcoxon Ranks Test) and between the different groups (ANCOVA) 9.2.1.2.1

Total variance of HRV

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test). Both groups showed directly (t1) a significant increase for the global HRV index (SDNN ms: Expi.: 20.95%, 80.77 ± 31.00, 95% CI [67.18 – 94.35], Z= -2.58, p .010; Sham: 23%, 78.09 ± 43.03, 95% CI [59.23 – 96.94], Z= -3.32, p .001), but could not be

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considered as an effect. An increased tendency was found for the experimental group for Total power ms2 (Expi.: FFT: 12.92%, 3490.47 ± 2756.15, 95% CI [2282.54 – 4698.41], Z= -1.64, p .100; Sham: FFT: 29.59%, 3404.74 ± 4172.08, 95% CI [1576.24 – 5233.23], Z= -1.38, p .167), while the AR analysis showed an increase significant effect for the Sham (Expi.: 9.97%, 3574.74 ± 2549.86, 95% CI [2457.21 – 4692.26], Z= -1.34, p .179; Sham: 35.41%, 3689.84 ± 4576.05, 95% CI [1684.30 – 5695.38], Z= 2.76, p .006). The experimental group exhibited a higher degree on the sympathetic cardiac complexity (ApEn: 0.66%) and a higher global autonomous variability (D2: 13.53%) (See D2 infra: 9.2.1.2.6 Cardiac complexity and regularity).

The experimental group showed immediate a small variation between the subjects for SDNN ms (CVg: 0.38), Total power ms2 (FFT: CVg: 0.79, AR: CVg: 0.71) and D2 (CVg: 0.49), compared with the baseline (Sham: SDNN ms: CVg: 0.55; Total power ms2: FFT: CVg: 1.23, AR: 1.24; D2: CVg: 0.60).

The experimental group exhibited on the short-term (t2) an decrease for the global HRV index (SDNN ms). A increased trend was found in the Sham group for: SDNN ms (Expi.: -3.88%, 64.19 ± 33.15, 95% CI [49.66 – 78.71], Z= -.34, p .737; Sham: 12.04%, 71.13 ± 32.68, 95% CI [56.81 – 85.45], Z= -1.61, p .108); Total power ms2 FFT (Expi.: 23.97%, 2349.97 ± 1802.89, 95% CI [1559.82 – 3140.12], Z= -.77, p .765; Sham: 24.97%, 3283.49 ± 3294.87, 95% CI [1839.45 – 4727.52], Z= -.14, p .135) and D2 (Expi.: -22.40%, 2.28 ± 1.83, 95% CI [1.48 – 3.08], Z= -.75, p. 455; Sham: 16.21%, 2.99 ± 1.58, 95% CI [2.30 – 3.68], Z= -1.61, p .108). The experimental group exhibited for SDNN ms on short-term a opposite between the Mean (decrease) and Median (increase), compared with the baseline. Based on the intra-individual differences, 60% of the subjects showed an increase in SDNN ms, compared with the individual baseline. The average actual change within-subjects (5.43%) and between-subjects (3.88%) gave ambiguity in the interpretation of an increase (within-subjects) or decrease (between-subjects) of SDNN. The sham showed for Total power ms2 on short-term a opposite between the Mean (decrease) and Median (increase), compared with the baseline. Based on the intra-individual differences, 50% (FFT analysis) and 60% (AR analysis) of the subjects showed an decrease in Total power ms2, compared with the individual baseline. The average actual change within-subjects (FFT: 63.91%, AR: 41.23%) and between-subjects (FFT: -23.97%, AR: -29.70) gave ambiguity in the interpretation of an increase (within-subjects) or decrease (between-subjects) of Total power ms2.

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The experimental group showed on the short-term a large variation between the subjects for SDNN ms (CVg: 0.52), D2 (CVg: 0.80) and a small variation for Total power ms2 (FFT: CVg: 0.77, AR: CVg: 0.76), compared with the baseline (Sham: SDNN ms: CVg: 0.46; Total power ms2: FFT: CVg: 1.00, AR: CVg: 1.04; D2: CVg: 0.53).

A significant difference was only found on the short-term between the different groups (ANCOVA) for: D2, with a large effect size: F (1,37)= 7.99, p .013, ηp2= .156, SDNN ms [K], with a medium effect size: F (1,37)= 4.55, p .040, ηp2=.109). A tendency was found for Total power ms2 t2 in both analyses, with a medium effect size (FFT: F (1,37)= 3.03, p .090, ηp2= . 076; AR: F (1,37)= 2.89, p .097, ηp2= .072).

9.2.1.2.2

Parasympathetic activity

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test). The Sham showed directly (t1) an increased significant effect on rMSSD ms (Expi.: 0.90%, 67.54 ± 40.69, 95% CI [49.70 – 85.37], Z= -1.16; p .247; Sham: 13.59%, 64.12 ± 48.64, 95% CI [42.80 – 85.44], Z= -2.28, p .023). The percentage ratio between the number of NN intervals (> 50 ms) was strongly increased at the experimental group (pNN50 % [K]: Expi.: 11.51%, 33.03 ± 19.30, 95% CI [24.57 – 41.48], Z= -1.31, p .191; Sham: 3.16%, 27.57 ± 21.49, 95% CI [18.15 – 36.99], Z= -.49, p .627). The Sham showed immediately for pNN50 % a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intraindividual differences, 55% of the subjects showed an increase in pNN50, compared with the individual baseline. The average actual change within-subjects (47.95%) and between-subjects (3.16%) confirm the increase that was seen in the arithmetic mean of pNN50. The Sham exhibited directly a significantly increased effect on the instantaneous beat-to-beat (short-term) variability (SD1 ms: Expi.: 6.19%, 44.85 ± 25.18, 95% CI [33.81 – 55.89], Z= -1.27, p .204; Sham: 8.78%, 41.60 ± 28.10, 95% CI [29.28 – 53.91], Z= -2.54, p .011). A direct increase of the cardiac vagal control (RSA: Expi.: 11.54%, 91.71 ± 45.21, 95% CI [71.90 – 111.53], Z= -1.20, p .232; Sham: 5.39%, 79.23 ± 38.62, 95% CI [62.31 – 96.16], Z= -1.49, p .135) with a reduction in the average heart rate was felt in both groups (Expi.: -2.55%, 63.52 ± 10.33, 95% CI [59.00 – 68.05], Z= -1.94, p .052; Sham: -1.06%, 62.17 ± 9.38, 95% CI [58.06- 66.28], Z= 1.08, p. 279). The Sham exhibited immediately for RSA a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intraindividual differences, 60% of the subjects showed an increase in RSA, compared with the individual baseline. The average actual change within-subjects (3.96%) and

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between-subjects (5.39%) confirm the increase that was seen in the arithmetic mean of RSA. The experimental group showed for average heart rate directly a opposite between the Mean (decrease) and Median (increase), compared with the baseline.

Based on the intra-individual differences, 70% of the subjects showed an decrease in the average heart rate, compared with the individual baseline. The average actual change within-subjects (-3.47%) and between-subjects (-2.55%) confirm the decrease that was seen in the arithmetic mean of the average heart rate. The intervention showed directly a reduction on the respiratory rate bpm (Expi.: -1.17%, 15.63 ± 2.68, 95% CI [14.45 – 16.80], Z= -.49, p .627; Sham: 0.12%, 16.02 ± 2.49, 95% CI [14.93 – 17.11], Z= -.08, p .940). The experimental group exhibited immediately for respiration rate bmp a opposite between the Mean (decrease) and Median (increase), compared with the baseline. Based on the intra-individual differences, 65% of the subjects showed an decrease in the respiration rate, compared with the individual baseline. The average actual change within-subjects (-5.94%) and between-subjects (-1.17%) confirm the decrease that was seen in the arithmetic mean of the respiration rate. The Sham showed for respiration rate bmp directly a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intra-individual differences, 55% of the subjects showed an decrease in the respiration rate, compared with the individual baseline. The average actual change within-subjects (3.84%) and between-subjects (0.12%) confirm the increase that was seen in the arithmetic mean of the respiration rate. Both groups exhibited in the frequency domain (FFT) analysis directly an increase in HF ms2 FFT (Expi.: 10.99%, 1438.54 ± 1180.65, 95% CI [921.09 – 1955.98], Z= -.86, p .391; Sham: 17.98%, 1414.92 ± 2005.29, 95% CI [536.06 – 2293.78], Z= -3.7, p .709). The experimental group showed an decrease for HF ms2 AR (-5.08%, 1625.89 ± 1327.26, 95% CI [1044.19 – 2207.59], Z= -1.08, p. 279), the opposite of the sham (21.59%, 1483.04 ± 1986.28, 95% CI [612.52 - 2353.57], Z= 1.49, p .135). A difference was seen in the experimental group for HF ms2 AR between the Mean (decrease) and Median (increase). Based on the intra-individual differences, 65% of the subjects showed directly an increase in HF ms2 AR, compared with the individual baseline. This is contradictory with the average actual change within-subjects (5.14%) and between-subjects (-5.08%). In the Sham group, the HF ms2 (FFT, AR) was different for the Mean (increase) and Median (decrease). Based on the intraindividual differences, 60% of the sham subjects showed directly an decrease of HF ms2 FFT while 65% showed an increase in the AR analysis, compared with the individual baseline. The average actual change within-subjects (FFT: 0.76%, AR: 1.57%) and between-subjects (FFT: 17.98%, AR: 21.59) confirm the increase that was seen in the arithmetic mean in both analysis for HF ms2. Both groups showed an

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decrease for HF n.u.FFT (Expi.: -5.50%, 47.12 ± 14.56, 95% CI [40.73 – 53.50], Z= 1.34, p .179; Sham: -7.72%, 42.44 ± 14.28, 95% CI [36.18 – 48.70], Z = -1.12, p .263.

The Sham exhibited immediate a significantly reduced effect on HF n.u. AR (Expi.: 7.36%, 46.09 ± 13.10, 95% CI [40.35 – 51.83], Z= -1.46, p .145; Sham: -8.99%, 41.41 ± 12.06, 95% CI [36.12 – 46.69], Z= -2.17, p .030).

The experimental group showed immediately a small variation between the subjects for: rMSSD ms (CVg: 0.60), pNN50 % (CVg: 0.58), SD1 ms (CVg: 0.56), RSA: (CVg: 0.49), respiration rate bpm (CVg: 0.17), HF ms2 FFT (CVg: 0.82) HF n.u. AR (CVg: 0.28) and a large variation for HF n.u.(FFT: CVg: 0.31), compared with the baseline (Sham: rMSSD ms [V]: CVg: 0.76; pNN50 %: CVg: 0.78; SD1 ms: CVg: 0.68; RSA: CVg: 0.49; respiration rate bpm: CVg: 0.16; HF ms2 FFT : CVg: 1.42; HF n.u.(FFT: CVg: 0.34, AR: CVg: 0.29)).

Both groups proved a short-term (t2) significance for rMSSD ms (Expi.: -35.09%, 43.44 ± 27.97, 95% CI [31.18 – 55.70], Z= -2.09, p .037; Sham: 11.89%; 63.16 ± 44.92, 95% CI [43.47 – 82.84], Z= -2.05, p .040) and pNN50 % (Expi.: -35.07%, 19.23 ± 19.03, 95% CI [10.89 – 27.57], Z= -2.35, p .019; Sham: 24.40%, 33.24 ± 25.79, 95% CI [21.94 – 44.55], Z= -2.43, p .015), but this could not be considered as effect. The intervention increase the instantaneous beat-to-beat RR-interval (short-term) variability (SD1 ms: Expi.: -30.65%, , 29.29 ± 17.33, 95% CI [21.69 – 36.88], Z= -1.98, p .048; Sham: 19.56%, 45.72 ± 28.71, 95% CI [33.13 – 58.30], Z= -2.58, p .010) and reduce the cardiac vagal control (RSA: Expi.: -20.07%, 65.72 ± 38.99, 95% CI [48.63 – 82.81], Z= -1.79, p .073; Sham: 10.13%, 82.80 ± 47.00, 95% CI [62.20 – 103.39], Z= -1.49, p .135). The Sham exhibited for RSA on short-term a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intraindividual differences, 75% of the subjects showed an increase in RSA, compared with the individual baseline. The average actual change within-subjects (10.53%) and between-subjects (10.13%) confirm the increase that was seen in the arithmetic mean of RSA. An increase on the respiratory frequency (bpm) was observed in both groups (Expi.: 5.06%, 16.61 ± 2.95, 95% CI [15.32- 17.90], Z= -1.27, p .204; Sham: 4.40%, 16.70 ± 2.40, 95% CI [15.65 – 17.76], Z= -1.08, p .279). The experimental group showed on the short-term an increasement of the average heart rate bmp [V] (5.29%, 68.63 ± 8.91, 95% CI [64.73 – 72.54], Z= -1.27, p .204). On the contrary, the Sham presented, depending on the used HRV software program (VU-DAMS, Kubios), an increasement or decrease of the average heart rate bpm ([V]: 1.71%, 63.90 ± 10.12, 95% CI [59.47- 68.34, Z= -.04, p .970; [K]: -0.14%, 62.76 ± 9.10, 95% CI [58.77 -

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66.75], Z= - .67, p .502). The experimental group showed on short-term a significant downward effect on the cardiac vagal control (HF ms2 FFT: Expi.: -44.02%, 725.51 ± 755.97, 95% CI [394.19 – 1056.82], Z= -2.09, p .037; Sham: 42.56%, 1709.76 ± 2421.97, 95% CI [648.28 – 2771.24], Z= -1.76, p .079). In both groups significance was assessed for HF ms2 AR (Expi.: -52.61%, 811.74 ± 843.82, 95% CI [441.92 – 1181.56], Z= -2.09, p .037; Sham: 51.38%, 1846.35 ± 2226.77], Z= -2.09, p .037), but couldn’t be considered as an effect. A significant reduction was also confirmed in the experimental group for HF n. U in both analysis (FFT: Expi.: -20.46%, 39.66 ± 17.44, 95% CI [32.01 – 47.30], Z= -2.39, p .017; Sham: 3.29%, 47.50 ± 17.73, 95% CI [39.73 – 55.27], Z= -.41, p .681; AR: Expi.: -19.94%, 39.83 ± 15.91, 95% CI [32.86 – 46.80], Z= -2.39, p .017; Sham: 4.47%, 47.53 ± 16.51, 95% CI [40.29 – 54.76], Z= -.14, p.

681). The Sham exhibited for HF n.u.ms2 FFT on short-term a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intra-individual differences, 50% of the subjects showed an increase in HF n.u.FFT, compared with the individual baseline. The average actual change within-subjects (5.35%) and between-subjects (3.29%) confirm the increase that was seen in the arithmetic mean of HF n.u.ms2 FFT.

The experimental group showed on short-term a small variation between the subjects for rMSSD ms (CVg: 0.64), SD1 ms (CVg: 0.59) and a large variation for pNN50 % (CVg: 0.99), RSA: (CVg: 0.59), HF n.u.(FFT: CVg: 0.44, AR: CVg: 0.40) an equal variation for respiration rate bpm (CVg: 0.18) and HF ms2 FFT (CVg: 1.04), compared with the baseline (Sham: rMSSD ms: CVg: 0.71; pNN50 %: CVg: 0.78; SD1 ms: CVg: 0.63; RSA: CVg: 0.57; respiration rate bmp: CVg: 0.14; HF ms2 FFT (CVg: 1.42); HF n.u.(FFT: CVg: 0.37, AR: 0.35)).

Only in the short-term (t2) there was a significant difference found between the different groups (ANCOVA) for: rMSSD ms, with a medium/large effect size (F(1,37)= 4.48, p .041, ηp2 = .108; [K]: F(1,37)= 8.39, p .006, ηp2= .185); pNN50 %, with a large effect size (F(1,37)= 11.12, p .002, ηp2= .231); HF ms2, with a large/medium effect size ([V]: FFT, F(1,37)= 6.85, p .013, ηp2= .156; [K]: FFT, F(1,37)= 4.58, p .039, ηp2 .110; [K] AR, F(1,37)= 5.70, p .022, ηp2= .133); HF n.u.AR, with a medium effect size ([K]: F(1,37)= 5.25,p .028, ηp2= .124); SD1 ms, with a large effect size (F(1,37)= 8.39, p .006, ηp2 = .185) and RSA, with a medium effect size (F(1,37)= 4.62, p .038, ηp2 = .111).

A tendency was noted for HF n.u.FFT t2, with a medium effect size (F (1,37)= 3.98, p .053, ηp2= .097).

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9.2.1.2.3

Sympathetic activity

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test) the experimental group showed directly (t1) an excitation of the sympathetic cardiac control, while this was inhibited at the Sham (PEP ms: Expi.: -2.43%, 88.43 ± 20.08, 95% CI [79.63 – 97.23], Z= -.49, p. 627; Sham: 4.43%, 94.56 ± 13.32, 95% CI [88.72 – 100.39], Z= -1.85, p .065). The experimental group showed for PEP ms directly a opposite between the Mean (decrease) and Median (increase), compared with the baseline. Based on the intra-individual differences, 60% of the subjects showed directly an increase, compared with the individual baseline. The average actual change within-subjects (-5.60%) and between-subjects (-2.43%) confirm the decrease that was seen in the arithmetic mean of PEP. Both groups showed a significant increase on the level of physiological arousal (SCL µS: Expi.: 22.90%, 5.23 ± 3.17, 95% CI [3.84 – 6.62], Z= -2.43, p .015; Sham: 45.74%, 4.75 ± 2.37, 95% CI [3.71 – 5.79], Z= 3.58, p <.001), but this couldn’t be considered as effect.

The experimental group showed immediate a large variation between the subjects for PEP ms (CVg: 0.23) and a small variation for SCL µS (CVg: 0.61), compared with the baseline (Sham: PEP ms: CVg: 0.14; SCL µS: CVg: 0.50).

In the short-term (t2), both groups exhibited an inhibition on the sympathetic cardiac control (PEP ms: Expi.: 4.36%, 94.59 ± 15.05, 95% CI [88.01 – 101.17], Z= -1.90, p .057; Sham: 1.34%, 91.76 ± 13.87, 95% CI [85.68 – 97.84], Z= -1.16, p .274), while the level of physiological arousel increased (SCL µS: Expi.: 18.55%, 5.05 ± 3.62, 95% CI [3.46 – 6.63], Z= -1.49, p .135; Sham: 18.34%, 3.86 ± 2.85, 95% CI [2.61-5.11], Z= .37, p. 709). In the Sham, PEP ms and SCL µS exhibited a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intraindividual differences, 70% of the subjects showed on short-term an increase in PEP, compared with the individual baseline. The average actual change within-subjects (2.36%) and between-subjects (1.34%) confirm the increase that was seen in the arithmetic mean for PEP. 50% of the subjects exhibited on short-term an increase in SCL. The average actual change within-subjects (23.81%) and between-subjects (18.34%) confirm the increase that was seen in the arithmetic mean for SCL.

The experimental group showed on short-term a small variation between the subjects for PEP ms (CVg: 0.16) and SCL µS (CVg: 0.72), compared with the baseline (Sham: PEP ms: CVg: 0.15; SCL µS: CVg: 0.74).

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Only a tendency was immediately (t1) found between the different groups (ANCOVA) for PEP ms with a medium effect size, F(1,37)= 3.86, p .057, ηp2= .095.

9.2.1.2.4

Sympathovagal balance

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test) the Sham exhibited directly (t1) a significant increased effect on the baroreceptors activity and between the mix of the ANS (LF ms2: Expi.: FFT: 42.44%, 1646.34 ± 1224.83, 95% CI [1109.53 – 2183.14], Z = -1.87, p. 062; AR: 27.89%, 1691.75 ± 1464.94, 95% CI [1149.94 – 2233.57], Z= -1.64, p .100; Sham: FFT: 33.50%, 1954.58 ± 2449.05, 95% CI [881.23 – 3027.92], Z= -2.69, p .007, AR: 42.71%, 1945.35 ± 2369.85, 95% CI [906.72 – 2983.99], Z= -2.69, p .007). Both groups showed directly an increase for LF n.u.FFT (Expi.: 5.50%, 52.81 ± 14.62, 95% CI [46.40 – 59.22], Z = -1.34, p .179; Sham: 6.67%, 57.50 ± 14.27, 95% CI [51.25 – 63.76], Z= -1.20, p .232). An significant increased effect was seen in the Sham group for LF n.u. AR (Expi.: 7.32%, 53.84 ± 13.15, 95% CI [48.07 – 59.61], Z= -1.46, p .145; Sham: 12.55%, 58.54 ± 12.06, 95% CI [53.25 – 63.82], Z= -2.17, p .030). Both groups showed directly an increased tendency in the autonomic cardiovascular regulation system (LF/HF ratio ms2: Expi.: FFT 7.46%, 1.35 ± 0.85, 95% CI [0.98 – 1.73], Z= 1.49, p .135; AR: 1.27%, 1.37 ± 0.81, 95% CI [1.02 – 1.73], Z= -.56, p .575; Sham: FFT: 14.18%, 1.75 ± 1.42, 95% CI [1.13 – 2.37], Z= -1.34, p .179; AR: 15.47%, 1.71 ± 1.15, 95% CI [1.21 – 2.22], Z= -1.61, p .108). The experimental group showed directly a reduction in the respiratory rate bpm, while it increased for the Sham (see supra: 9.2.1.2.2).

The experimental group showed immediate a small variation between the subjects for LF ms2 (FFT: CVg: 0.74, AR: CVg: 0.73), LF n.u.(FFT: 0.28, AR: CVg: 0.24) and LF/HF ratio ms2 (FFT: CVg: 0.63, AR: CVg: 0.59), compared with the baseline (Sham: LF ms2 FFT: CVg: 1.25, AR: CVg: 1.22; LF n. U.: FFT: CVg: 0.25, AR: CVg: 0.21; LF/HF ratio ms2: FFT: CVg: 0.81, AR: CVg: 0.67).

The experimental group and sham showed on the short-term (t2) an decrease or increase for LF ms2 FFT (Expi.: -6.67%, 1078.80 ± 910.32, 95% CI [679.83 – 1477.76], Z = -.075, p .940; Sham: -5.61%, 1381.91 ± 1402.97, 95% CI [767.04 – 1996.79], Z= .971, p .332). An opposite was seen in the experimental group for LF ms2 FFT between the Mean (decrease) and Median (increase), compared with the baseline. Based on the intra-individual differences, 50% of the experimental subjects showed an decrease in

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LF ms2 FFT, compared with the individual baseline. This is contradictory with the average actual change within-subjects (29.41%) and between-subjects (-6.67%). At the Sham, 65% of the subjects showed an decrease in LF ms2 FFT, compared with the individual baseline. This is contradictory with the average actual change within-subjects (40.94%) and between-subjects (-5.61%). The experimental group showed an decrease for LF ms2 AR (Expi.: -14.16%, 998.97 ± 963.43, 95% CI [707.32 – 1551.81], Z= -.30, p .765), while it was increased in the Sham (5.42%, 1437.11 ± 1416.90, 95% CI [816.12 – 2058.09], Z= -1.12, p .263). An opposite was observed in the experimental group for LF ms2 AR between the Mean (decrease) and Median (increase). Based on the intra-individual differences, 55% of the experimental subjects showed on short-term an decrease in LF ms2 AR , compared with the individual baseline. This is contradictory with the average actual change within-subjects (21.00%) and between-subjects (14.61%). The experimental group showed on short-term a significantly increased effect on LF n.u.(Expi.: FFT: 20.42%, 60.28 ± 17.45, 95% CI [52.63 – 67.93], Z= -2.39, p .017, AR: 19.80%, 60.10 ± 15.94, 95% CI [53.12 – 67.09], Z= -2.39, p .017; Sham: FFT: -2.75%, 52.42 ± 17.73, 95% CI [44.65 – 60.19], Z= -.41, p .681, AR: 0.75%, 52.40 ± 16.52, 95% CI [45.16 – 59.64], Z= -.78, p .433). The Sham exhibited for LF n.u.(FFT, AR) a opposite between the Mean (decrease) and Median (increase). Based on the intra-individual differences, 50% of the subjects showed on short-term an decrease in LF n. U., compared with the individual baseline. The average actual change withinsubjects (-0.24%) and between-subjects (-2.75%) confirm the decrease that was seen in the arithmetic mean of LF n.u.FFT. 55% of the subjects showed on short-term an increase in LF n.u.AR, compared with the individual baseline. This is contradictory with the average actual change within-subjects (-0.81%) and between-subjects (0.75%). On the short-term the experimental group showed a significantly increased effect on the autonomic cardiovascular regulation system (LF/HF ratio ms2 FFT: Expi: [K]: 64.37%, 2.07 ± 1.39, 95% CI [1.46 – 2.68], Z= -2.17, p .030; [V]: 2.21 ± 1.57, 95% CI [1.52 – 2.90], Z= -2.43, p .015; Sham: [K]: -0.79%, 1.52 ± 1.36, 95% CI [0.92 – 2.12], Z= -.15, p .881, [V]: 1.66 ± 1.52, 95% CI [0.99 – 2.32], Z= -.41, p .681).

The experimental group showed on short-term a small variation between the subjects for: LF ms2 (FFT: CVg: 0.84, AR: CVg: 0.85), LF n.u.(FFT: CVg: 0.29, AR: CVg: 0.27) and LF/HF ratio ms2 (FFT: CVg: 0.67, AR: CVg: 0.67), compared with the baseline (Sham: LF ms2 FFT: CVg: 1.02; AR: CVg: 0.99, LF n. U.: FFT: CVg: 0.34, AR: CVg: 0.32, LF/HF ratio [K]: FFT: CVg: 0.90, AR: CVg: 0.91).

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Significance was only found on the short-term (t2) between the different groups (ANCOVA) for: LF n.u.AR, with a medium effect size F(1,37)= 5.27, p .028, ηp2= .125); LF/HF ratio ms2 FFT, with a medium effect size F(1,37)= 4.44, p .042, ηp2= .107).

Tendencies were noted for: LF n.u.FFT t2, with a medium effect size F(1,37)= 3.97, p .054, ηp2= .097); LF/HF ratio FFT t2, with a medium effect size F(1,37) = 3.56, p .067, ηp2= .088; LF/HF ratio AR t2, with a medium effect size F(1,37) = 2.94, p .095, ηp2= .074).

9.2.1.2.5

Scaling behavior of the ANS

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test) both groups exhibited in the scaling behavior of the ANS directly (t1) an increase on: the short-term fluctuations (DFA α1: Expi.: 2.50%, 0.98 ± 0.21, 95% CI [0.88 – 1.07], Z = -.60, p .550; Sham: 5.52%, 1.02 ± 0.18, 95% CI [0.94 – 1.10], Z= -1.68, p .093), the long-term fluctuations (DFA α2: Expi.: 30.06%, 0.32 ± 0.16,95% CI [0.25 – 0.39], Z= -2.17, p .030; Sham: 13.59%, 0.29 ± 0.11, 95% CI [0.24 – 0.34], Z= -1.90, p .057) and on the long-term variability (SD2 ms: Expi.: 13.05%, 68.01 ± 27.14, 95% CI [56.11 – 79.90], Z= -179, p .073; Sham: 16.07%, 66.74 ± 36.35, 95% CI [50.81 – 82.68], Z= -2.76, p .006).

The experimental group showed immediate a small variation between the subjects for: DFA α1 (CVg: 0.22), SD2 ms (CVg: 0.40) and a large variation for DFA α2 (CVg: 0.51), compared with the baseline (Sham: DFA: α1: CVg: 0.18, α2: CVg: 0.39; SD2 ms: CVg: 0.54).

At the short-term (t2), the experimental group exhibited an increased tendency for the short-term fluctuations (DFA α1: Expi.: 11.56%, 1.06 ± 0.22, 95% CI [0.97 – 1.16], Z= 1.68, p .093; Sham: -4.50%, 0.92 ± 0.29, 95% CI [0.80 – 1.05], Z= -.90, p .370). The experimental group showed a significantly increased effect on the long-term fluctuations (DFA α2: Expi.: 49.23%, 0.37 ± 0.11, 95% CI [0.32 – 0.41], Z= -3.80, p < .001; Sham: 3.44%, 0.27 ± 0.12, 95% CI [0.21 – 0.32], Z= -.49, p .627). The Sham exhibited for DFA α2 a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intra-individual differences, 55% of the subjects showed on short-term an decrease in DFA α2, compared with the individual baseline. The average actual change within-subjects (8.41%) and between-subjects (3.44%) confirm the increase that was seen in the arithmetic mean of DFA α2. The long-term variability was reduced at the experimental group, while this significantly

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increased for the Sham (SD2 ms: Expi.: -11.43%, 53.28 ± 24.24, 95% CI [42.65 – 63.90], Z= -.71, p .478; Sham: 9.29%, 62.84 ± 26.09, 95% CI [51.41 – 74.28], Z= -2.76, p .006).

The experimental group showed on short-term a small variation between the subjects for: DFA (α1: CVg: 0.20; α2: CVg: 0.31), SD2 ms (CVg: 0.45), compared with the baseline (Sham: DFA: α1: CVg: 0.32, α2: CVg: 0.44; SD2 ms: CVg: 0.42).

Significance was only found for short-term (t2) between the different groups (ANCOVA) for: SD1 ms, with a large effect size F(1,37)= 8.39, p .006, ηp2= .185), DFA α1, with a medium effect size F(1,37)= 4.93, p .033, ηp2= .117), DFA α2, with a large effect size F(1,37)= 19.16, p < .001, ηp2= .341). Tendency was found for SD2 t2, with a medium effect size F(1,37)= 2.38, p .132, ηp2= .060).

9.2.1.2.6

Cardiac complexity and regularity

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test) the experimental group exhibited directly (t1) an increase in the complexity and regularity of the physiological time series, the opposite at the Sham (ApEn: Expi.: 0.66%, 1.29 ± 0.09, 95% CI [1.25 – 1.32], Z= -.41, p .681; Sham: -0.09%, 1.30 ± 0.12, 95% CI [1.25 -1.36], Z= -.37, p .709). In both groups ShanEntropy was increased: (Expi.: 1.11%, 3.10 ± 0.26, 95% CI [2.99 – 3.22], Z= -1.20, p. 232; Sham: 2.96%, 3.11 ± 0.26, 95% CI [2.99 – 3.22], Z= -2.20, p .028). The experimental group exhibited immediately a stronger inhibition on the sympathetic cardiac complexity (D2: Expi.: 13.61%, 3.34 ± 1.62, 95% CI [2.63 – 4.04], Z= -.75, p .455; Sham:1.94%, 2.63 ± 1.59, 95% CI [1.93 – 3.32], Z= -1.61, p .108). The Sham group showed immediately for D2 a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intra-individual differences, 60% of the subjects showed an decrease in D2, compared with the individual baseline. The average actual change within-subjects (741.65%) and between-subjects (2.01%) showed an increase in D2.

The experimental group showed immediate a small variation between the subjects for: ApEn (CVg: 0.07), ShanEntropy (CVg: 0.08), D2 (CVg: 0.49) compared with the baseline (Sham: ApEn: CVg: 0.09; ShanEntropy: CVg: 0.08; D2: CVg: 0.60).

The experimental group showed on the short-term (t2) a significantly increased effect on the cardiac complexity and regularity of the physiological time series (ApEn: Expi.:

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4.06%, 1.33 ± 0.10, 95% CI [1.29 – 1.37], Z= -2.24, p .025; Sham: 1.53%, 1.33 ± 0.08, 95% CI[1.29 – 1.36],Z= -.64, p .526). The Sham exhibited for ApEn on short-term a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intra-individual differences, 50% of the subjects showed an increase in ApEn, compared with the individual baseline. The average actual change within-subjects (2.32%) and between-subjects (1.68%) confirm the increase that was seen in the arithmetic mean of ApEn. The distribution of physiological patterns were increased at the experimental group, while this reduces at the Sham (ShanEntropy: Expi.: 1.19%, 3.11 ± 0.21, 95% CI [3.01 – 3.20), Z= -.26, p .794; Sham: - 0.89%, 2.99 ± 0.26, 95% CI [2.88 – 3.11], Z= -.67, p .502). The Sham exhibited for ShanEntropy on short-term a opposite between the Mean (decrease) and Median (increase), compared with the baseline. Based on the intra-individual differences, 60% of the subjects showed an decrease in ShanEntropy, compared with the individual baseline. The average actual change within-subjects (-0.43%) and between-subjects (-0.89%) confirm the decrease that was seen in the arithmetic mean of ShanEntropy . A reduced degree of complexity in the ANS was seen in the experimental group, the opposite of the Sham (D2: Expi.: -22.40%, 2.28 ± 1.83, 95% CI [1.48 – 3.08], Z= -.75, p. 455; Sham: 16.21%, 2.99 ± 1.58, 95% CI [2.30 – 3.68], Z= -1.61, p .108). The experimental group showed a reduction for D2 in the Mean and Median, compared with the baseline.

Based on the intra-individual differences, 60% of the subjects showed an increase in D2, compared with the individual baseline. The average actual change within-subjects (0.87%) and between-subjects (-22.40%) gave ambiguity in the interpretation of an increase (within-subjects) or decrease (between-subjects) of D2.

The experimental group showed on short-term a small variation between the subjects for: ApEn (CVg: 0.07), ShanEntropy (CVg: 0.07) and a large variation for D2 (CVg: 0.80), compared with the baseline (Sham: ApEn: CVg: 0.06; ShanEntropy : CVg: 0.09; D2: CVg: 0.53).

Significance was only found for short-term (t2) between the different groups (ANCOVA) for D2, with a large effect size F(1,37)= 6.86, p .013, ηp2 = .156.

9.2.1.2.7

Blood Pressure

Compared to the baseline in the same group (Wilcoxon Ranks Test) the intervention (based on the Mean) exhibited directly (t1) an increase on the systolic and diastolic blood pressure (right), while it was decreased in the Sham (SBPR mm Hg: Expi.:

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0.09%; 117.10 ± 14.56, 95% CI [110.72 – 123.48], Z= -.36, p .722; Sham: -1.49%, 119.15 ± 18.85, 95% CI [110.89 – 127.41], Z= -1.08, p .279; DBPR mm Hg: Expi.: 1.42%, 75.10 ± 9.97, 95% CI [70.73 – 79.47], Z= -.65, p .519; Sham: -1.77%, 77.70 ± 9.96, 95% CI [73.33- 82.07], Z= -.84, p .400). The experimental group exhibited immediately for DBPR a opposite between the Mean (increase) and Median (decrease), compared with the baseline. Based on the intra-individual differences, 55% of the subjects showed an increase in DBPR, compared with the individual baseline.

The average actual change within-subjects (1.28%) and between-subjects (1.42%) confirm the increase that was seen in the arithmetic mean of DBPR.

The experimental group showed immediately a large variation between the subjects for SBPR (CVg: 0.12) and DBPR (CVg:0.13), compared with the baseline (Sham: SBPR mm Hg: CVg: 0.16; DBPR mm Hg: CVg:0.13).

On the short-term (t2), the Sham showed a significant decreased effect on the systolic and diastolic blood pressure (SBPR mm Hg: Expi.: -3.38%, 113.05 ± 12.04, 95% CI [107.77 – 118.33], Z= -1.88, p .061; Sham: -6,24%, 113.40 ± 10.14, 95% CI [108.95 – 117.85], Z= -2.58, p .010; DBPR mm Hg: Expi.: -3.31%, 71.60 ± 8.70, 95% CI [67.79 – 75.41], Z= -1.53, p .126; Sham: -3.67%, 76.20 ± 9.60, 95% CI [71.99 – 80.41], Z=2.021, p .043).

The experimental group showed on short-term a large variation between the subjects for SBPR mm Hg (CVg: 0.11) and DBPR mm Hg (CVg:0.12) (Sham: SBPR mm Hg: CVg: 0.09; DBPR mm Hg: CVg:0.13).

There was no significant difference found directly or on short-term between the different groups (ANCOVA) to SBPR and DBPR. Tendency was found for SBPL t1 with a medium effect size F(1,37) = 2.73, p = .107, ηp2 = .069.

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10 Part VII: Discussion The Belgian Osteopathic studies consulted with regard to HRV are not in accordance with the European and Belgian legislation (De Cock, 2015). A final favorable opinion of a CME is missing, as well as a flawless civil liability insurance. The methodological standard guidelines of the Task Force to obtain HRV parameters were not consistently respected.

Contradictions by common HRV parameters between software program VU-DAMS and Kubios were found concerning the significance for both tests.

The techniques used in both groups exhibited no side effects (adverse events), both immediately and in the short-term. Only local skin irritations of limited duration were observed in both groups after removing the ECG/ICG skin electrodes, but do not belong to side effects related to the applied techniques.

10.1 Discussion intent of RILHOU-RCT The practice-based evidence of the RILHOU-RCT can serve to formulate statements about the efficiency (under optimal conditions) and effectiveness (field conditions, pragmatic = total study) of the applied techniques on blood pressure, HRV, RSA, PEP, SCL, blood pressure on mentally competent Dutch-speaking adults between 20 and 50 years with a BMI ≤ 30 (except for one part of the research). 

In the RILHOU-RCT several research questions can be posed. For this study the specific research question is if the applied techniques (under optimal conditions) have a (treatment) effect on the above parameters. For the total investigation, the research question is focused on the effectiveness of indirect osteopathic techniques (not under optimal conditions) on the above parameters.



A general challenge is to enshrine and incorporate the research results in a common practice. This study possibly could contribute to the drive for greater efficiency and effectiveness (Total RILHOU-RCT) in scientific research within osteopathy (according to ICH-GCP guidelines, European and national legislation, correct methodology).

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

The design of the RILHOU-RCT can serve as a basic model or blueprint for further scientific research within osteopathy related to the ANS (e. G.: the reproduction of the study, drawing up standard values for the studied sample, drawing up possible explanatory models on the basic of the mediators "how it works", the use of the same methodology applied to various pathologies, etc.).



The study was not sponsored, hence no expectations could be imposed by a sponsor. Strictly scientific researchers should distance themselves from any expectation (De Cock, 2014).



Potential benefits associated with a (non-sponsored) line of research carried executed by several investigators: financial load versus research spirit, scientific coordination for the redirection of a study (according to ICH-GCP guidelines, European and national legislation), correct methodology, larger success rate to obtain a final favorable opinion by an independent CME (full recognized), quality control, logistic support in the design and implementation of the study. From a scientific point of view would a study (according to ICH-GCP guidelines and legislation) within osteopathy could mean that study results could be implemented in daily practice. Further, the study could be published in various professional journals, trainings or incorporated in the curriculum of osteopathy. A well-founded basic research makes follow-up studies possible.



Potential disadvantages associated with a (non-sponsored) line of research: the scientific coordination and management of the trial is time-consuming and financially (almost) impossible. In Belgium, grants or scholarships are no awarded for scientific research within osteopathy. Students themselves bear the responsible for the design and financing of a scientific investigation. The development of a protocol of good quality requires a lot of time and a lot of clinical experience and imagination of the student (De Cock,1998). The question whether a student can possess sufficient research experience to design a study protocol (-design) for an ideal clinical trial can be posed. A promoter can behave as a student guide, provided that he has adequate competences and research experience (De Cock, 2016).

The length of time for developing a study design is generally longer compared to an individual research because more parties are involved, e. G. to obtain a consensus, drafting the request for the CME. ... Further, the student is supposed to

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be able to work together in a group where the payload and responsibilities are proportional divided and included in benefit of the study. 

Osteopathic schools can play a fundamental role as ordering party and executor of scientific research within osteopathy (according to ICH-GCP guidelines and legislation), in which students can participate. Scientific research within osteopathy could, by this means, be able to offer relevant significance in the practice-based evidence science. Dissemination and valorization of the research results can therefore be implemented.

10.2 Discussion

European

and

Belgian

legislation

on

experiment on the human person.

The Nuremberg Code describes ten basic ethical principles to which an experiment need to comply. One basic principle of this code "experiments on people must be based on prior studies" could for this study not be entirely met. 

In cranial osteopathy empirical research is already done on animals including: the mobility of the os parietal of squirrel monkeys with a possible relationship between heart activity, respiratory system and other physiological events (Tiller et al. 1996; Ferguson et al. 1998), cats, goats (Chila, 2010); the effect of a cranial elevator technique on the skull of rabbits on the intracranial pressure and cranial bone 'movement' (Downey et al. 2006). The set of techniques in this study weren’t first subject on animals, which contest the basic ethical principles of the Nuremberg Code.

The main ICH-GCP guidelines can be subsumed under fourteen aspects and are aimed towards clinical trials with pharmaceutical purposes. The RILHOU-RCT does not use drug research, but has the same directives implemented in its methodology. Two aspects of these guidelines could not be met for the RILHOU-RCT. 

The RILHOU-RCT has no direct connection with a doctor or dentist. This implied that the medical care and medical decisions (for the benefit of the test subject) were not under sole responsibility of a doctor or dentist (i. E. a principal investigator). Before the start of this study the subjects could communicate their participation to their family doctor on the basis of an additional form specifically addressed to the family doctor.

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The legislative framework in Belgium on experiments on the human person is described in the law of 2004. This law includes 25 chapters and is politically subdivided into 36 articles. Here the legal guidelines for the application of clinical and medical trials for humans are described. The large part of the articles has more connection to pharmaceutical research. 

In chapter I: "general clause" describes the exercise of health care professions in accordance with the Royal Decree n° 78 of 10 November 1967 (B. S. 18 May 2004). For the regular exercise of non-conventional practices in medicine including osteopathy, reference is made to the law of 29 April 1999 (law Colla). The act of legally exercising health professions without a prescription on the domain 'medical' is a violation of the Royal Decree n° 78 (RvV, 2007). Osteopathy can legally one be performed when one is registered with one of the six Osteopathic professional organizations which have a recognition obtained by the Council of State (article 8. §1, law of 29 April 1999). To be fully able to be registered at one of the recognized professional associations one should hold: D. O. or BSc. or MSc. in osteopathy.

The title 'Doctor of Osteopathy (DO or D. O.) is only granted to fully qualified doctors in the USA (Gevitz, 2014). To obtain a degree in osteopathy the student is asked to write a scientific dissertation through conducting a scientific osteopathic study. When a student doesn’t hold a D. O. he cannot fully register with one of the Belgian recognized professional associations. As a result, a student in osteopathy doesn’t comply to article 8. §1 (law of 29 April 1999). Therefore there is no legal registration regarding to the execution of the non-conventional practice 'osteopathy', whereby a student does not comply with article 1. §1 (law of 7 May 2004). Accredited Belgian osteopathic associations can play an active role by, for example, including an additional clause in their policies with conditions for last year osteopathic students about the registration. In this way a student can meet the statutory provision in accordance with article 8. §1 (law of 29 April 1999) and article 1. §1 (law of 7 May 2004).



The investigation file in chapter II is described as a set of clinical and non-clinical data, but only relates only to drug research (article 2. 3, B. S. 18 May 2004). Each subject in the RILHOU-RCT has an extensive investigation file. The law includes no specific statement on the requirement for an investigation file in non-drugs research, but that doesn't mean that this directive can’t be applied for non-drugs research.

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

In Belgium a 'definitive' favorable opinion given out by a recognized CME is a legal obligation to perform an (interventional) experiment on human beings (article 5. 6°, B. S. 18 May 2004; Dresse, 2005). Without the exception of the free University of Brussels, where a complementary master in osteopathy can be obtained at the Faculty of human movement Sciences, there is no University in Belgium that organize Osteopathic training. As a result, a osteopathic student may not appeal to a local CME and should the request for advice for an experiment within osteopathy be directed to a fully recognized CME.



In the questions prepared by the CME ZOL the competence of the researchers and employees was questioned. In the reply, it was noted that students in osteopathy, physiotherapy, manual therapy, nursing, psychology, ... should make a dissertation that should be publicly defended for an international jury. Neither of these

students

has

research

experience.

In

a

university

setting

the

promoter/associate supervisor or supervisor (who doesn’t serve as researcher) will coordinate, adjust and direct the research (De Cock, 2014). A student is not qualified enough for the CME for a trial because there is no or insufficient research experience, neither a qualification. This argument can already be enough for a CME to provide a negative opinion on a request for advice. In Belgium, the legally performance of an (interventional) experiment on humans within osteopathy is almost impossible for students. 

The RILHOU-RCT (Eudract/B-nr. B371201422023) has received on 9 December 2014 by the CME ZOL a final approval for the period from 15 December 2014 – December 15, 2015. A provision for the period from 1 January 2016 – 31 December 2016 was obtained after submitting a petition to the CME ZOL. The extension period does not close completely with the previous validity period (December 15, 2014 – 15 December 2015) whereby there is a grey area between the two periods.



Insurance company Ethias extended free of charge the insurance flawless civil liability... following the previous validity period.



Article 2. 5 ° (B. S. 18 May 2004) refers to different definitions about side effects: unexpected side effects, adverse event, serious adverse event, and serious side effects. On the exception of 'adverse event' all other side effects are associated with trials which involves drugs.

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

In medical literature one can find different definitions about adverse reactions and events of medicinal interventions (Goldfarb, 2012). A common unanimity in the terminology is missing (Vogel, 2012). In the RILHOU-RCT were adverse events noted as ‘adverse events’.



The law provides that each study demands an ordering party (“sponsor”, person, company, institution, organization) who holds the responsibility on various aspects (article 2. 21° B. S. 18 May 2004) and can be held flawless liable for the damage sustained a test subject (article 29. §1, B. S. 18 May 2004). Since the RILHOURCT consist of seven ordering party’s it became impossible to take out a faultless insurance civil liability (article: 2. 21° and 29. §1, B. S. 18 May 2004). A solution was found by recognizing VZW Health as actual ordering party (“sponsor”) for the study. A request to obtain the legally required flawless civil liability for this study was immediately denied by four insurance companies (Ethias, Assura Portima, AMMA, Belfius Insurance).

Ethias mentioned that they initially could not provide for the compulsory insurance RILHOU-RCT. The reason of Ethias was stated as follow: "The term 'ordering party'.... The provided documents show that this is not CME-ZOL. The fact that the study was approved by the CME ZOL is irrelevant in this matter. Who the ordering party exactly is not completely clear. In our view, this is either 'VZW Health' or 'FICO'. It is therefore up to either this association or FICO to insurance the objective of civil liability. We grand this insurance only if the general professional liability of the association or institution has been endorsed. For this reason we recommend that you apply to the insurer of either VZW Health either FICO". In this correspondence it is specifically defined that the study already was approved by the CME ZOL, despite the request for advice for the RILHOU- RCT was not yet handled by the CME. The conditions that were mandatory to Ethias are not an endorsement of a CME, but that a general endorsed professional liability of either the association (vzw Health) or institution (Fico) is signed at Ethias.

Insurance company 'AMMA' still hasn’t offered a written confirmation of the negative opinion for the study. Belfius Insurance mentioned to Assura Portima that after performing a risk analysis of the RILHOU-RCT no proposal could be transferred: "we wish not to insure this risk for the following reason: nature of activities, liability issues, reinsurance problems".

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After active consultation with the contact person of the RILHOU-RCT at Ethias the legally compulsory insurance (policy number 45.326.306) could be granted. 

Although the RILHOU-RCT is classified under Phase 1 trials of 'initial efficacy measurement’ (article 2.12 °) this terminology is inconsistent with the provision of article 2. 7°, because here is only referred to clinical, pharmacological and/or other pharmacodynamics effects of medicines.

10.3 Discussion methodology The RILHOU-RCT belongs to a monocentric interventional trial, therefore the request was directed to a CME with full recognition (CME ZOL). The study has obtained a completely final favourable opinion for a two arms parallel study. A flawless civil liability insurance concerning experiments on the human person was completed for the total study. Only for researcher Wens C an additional clause "thesis Osteopathy to FICO" was included in his individual insurance policy civil liability. The RILHOU-RCT belongs to quantitative research because as much as possible of the research already was determined in advance (Aertgeerts and Buntinx, 2006). Before the initiation of the study all documents in the form of a 'Common documents' were legal deposited (De Cock, 2014). 

For the CME ZOL it the first time that they were confronted with a request for advice for a study in osteopathy. The CME questions whether if they have the competence to assess a study in osteopathy.



The questions and comments of the CME ZOL were not always clearly formulated whereby multiple interpretations were possible . The reply was largely conducted by the Scientific Coordinator (De Cock, 2014).



According to the remarks of the CME ZOL it is irrelevant to perform a study within osteopathy with regard to the autonomic nervous system with a much larger trial group since there was already sufficient research carried out in the past. In 2008, the study Vander Mast H. the null hypothesis does not confirm. The CME argues that in other studies a reverse hypothesis H1 would obtain. The null hypothesis (H0) in the thesis was, however, rejected, making the alternative hypothesis (H1) possible. The author refers that further investigation is required because of the limited sample test (Vander Mast H, 2008).

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

Some comments can be made on the thesis by Vander Mast on legislation and methodology to obtain HRV. The data of the ICF show that the research carried out in Belgium was "Done at Brasschaat on .../.../2007". Each experiment that is carried out in Belgium must comply with the legal obligations as referred to in the law of 7 May 2004 (B. S. 18 May 2004). The experiment was not subjected to a CME (with full recognition), whereby the study has obtained no definitive favorable opinion for it can conduct the experiment, which is a breach of article 5. 6°. The study has no insurance flawless civil liability on experiments on the human person, which is an infringement on article 29. §1. The subjects were informed through an ICF and gave their consent in writing. The ICF describes briefly the nature of the techniques (soft techniques on the skull, no manipulations), the objective (increase in HRV) and a consequence (may affect emotional system), the ability to force the investigation, contact details of the researcher. In the ICF is missing: the scope, the expectations, the risks of the experiment, the circumstances in which the experiment takes place, insurance technical aspects, the identification and the opinion of the competent CME (article 6. §1, B. S. 18 May 2004).

The inclusion of the R-R intervals was obtained via a EmWave (biofeedback device) and a Polar heart rate monitor (watch), which is not in conformity with the general guidelines of the Task Force 1996 to obtain HRV. 

In the original request for advice a three arms study (experimental group, Sham group, control group) was envisioned, but was, by suggestion of the CME ZOL, reduced to a two arms parallel study. The advantage of this study design (experimental group, Sham group) reduced the sample to fewer subjects since each group serves as own control group. The disadvantage of this study design is that no pure control group (nocebo) is present to investigate the experimental techniques and Sham with the experimental process. Therefore, the temporal stability and reliability can’t be guaranteed (De Cock, 2016).



The legal deposit of the Common documents gives the possibility that all information is publicly available so that the treatment can be checked (operandi) and external monitoring is possible.



In the ICF the test subject was not explicitly informed of a possible Sham approach. From an ethical point of view the mentioning of a Sham is absolutely correct. By not explicitly mentioning the subject doesn’t know the difference in

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between the various treatment modalities. Patients blinding was therefore not raised (De Cock, 1989). By this means a possible preferential treatment of the test person is eliminated. The question whether or not to explicitly mention a Sham in the ICF was submitted to the CME ZOL, but remained unanswered (De Cock, 2014).

For this study a FMEA was applied with the aim of identifying potential risks related to the conduct of the trial. Security measures were applied and processed in a separate working document RILHOU-RCT roadmap and FMEA 

The FMEA was checked and adjusted by a professional external security and prevention Advisor (Janssens P). The RILHOU-RCT roadmap and FMEA (working document) was applied to the whole study. The absence of the active participation of other researchers and scientific coordinator gives a deficit in the FMEA and the working document. For this study only a proactive analysis was work out, a retroactive analysis is missing.

Before the start of this study a small preliminary study was conducted. 

After the final approval of the CME minimal changes were made to the protocol (deviations) before the start of the study, which led to minor adjustments in the working document. These amendments were reported to the scientific coordinator.

The minimal changes did not affect the protocol whereby there are no protocol violations.

The study population consists of mentally competent Dutch-speaking adults between 20 and 50 years with a BMI ≤ 30. 

In literature the concept of 'health' is not unambiguously defined (Huber, 2014; Law and Wakame, 2008; Carel, 2009; Anger et al. 2013; Bircher, 2005). For example: the WHO defines ‘health’ as a state of complete physical, mental and social wellbeing and not merely the absence of diseases or disabilities (At: http://www. Who. Int/about/definition/en/print. Html). Huber et al. describe 'health' as 'the ability to adapt and a privately manage in the light of physical, emotional and social challenges of life' (Huber et al. 2011). The questionnaire lacks the specific question about the mental state of the social well-being of the test subject. It was tried as much as possible to exclude as much confounders relating to HRV. External

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variables such as room temperature, relative humidity percentage and environmental noise were recorded during the study. 

The survey was deliberately investigated with subjects between 20 and 50 years old because this population was the most obvious for the researcher. Pregnant women were excluded in order to raise the success rate for obtaining a favorable opinion of the CME, and retaining the homogeneity of the groups.

Subjects were recruited through flyers and social media. Most of the subjects came from province of Antwerp and had a direct or indirect relationship with the researcher. The sample is too limited to formulate generalized statements about Dutch-speaking adults between 20 and 50 years (Sutherland et al. 2013). To perform a study through an independent study 'selection and search company’ would be a solution for this problem (e. G.: At: https://www. Medians. Be/).

Regarding the financial limit of the study an independent 'selection and search firm' was not eligible. 

The test measurements were acquired via short-term registration under clinical conditions, which may already have an influence on the results. An ambulatory measurement of a long-term (24 h) gives a better prediction of the HRV values (Dikecligil and Mujica-Parodi, 2010). One VU-AMS device was available, therefore a long-term registration was impossible.



The blood pressure measurement was performed in another room than there where the room temperature was registered. Since the registration of the ECG, ICG and SCL (via the VU-AMS device) was performed in supine position, it had been better to perform the blood pressure registration in the same room and in supine position.

Some comments related to statistics: 

In the ANCOVA only the ‘control measurement' was anayzed as co-variation.

Analyzing the respiration rate as co-variance would have been an added value.

Breathing has an impact on HRV and RSA (Sin and Tzeng, 2010). Statistical subanalysis with stratification such as gender, age, BMI, and smoking were not performed before both. A description of the distribution of sociodemographic data was performed in Microsoft Office Excel 2007 for: nationality, gender, age, BMI, marital status, number of hours sports, shift patterns, smoking, number of glasses

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of alcohol a week, allergy, categorical scale related to the experience of pain, use of

non-conventional

medicine/or

alternative

form

omega

3

fatty

acids,

supplementation, medication, room temperature and relative humidity percentage (Addendum 8). 

The statistical review of the hypothesis with Alpha = 5% (p < 0.05) was used. The justification that Alpha 5% can be considered as an index or as consideration for the type I and II error is subjected to debate. This review is often wrongly interpreted as a measure for the extent of a found difference or practical relevance (Nuzzo, 2014; Wierdsma et al. 2013; Goodman, 1999; De Cock, 1989). A p-value of < 0.05 represents a 95% chance that a replication study would have the same outcome. Therefore the assumption that the null hypothesis is correct and rejected by a small p-value (p < 0.05) is not accurate to interpret non-significant results as no difference or no effect (Walford et al. 2013). The probability (95%), however, is a choice that in sociology, psychology, economics, epidemiology, medical and statistical criteria is up for discussion (Wierdsma et al. 2013; De Cock, 1989).



In total, the RILHOU-RCT exists of seven studies , which enables the possibility of a meta-analysis. Research has show, that when small studies are being made available for a meta-analysis (even when subject to publication bias and heterogeneity) this results in lower rates of false-positive findings, in opposition to a new and larger trial (in 't Hout, 2015).



The confidence interval (interval approximately) to point the average differences between both groups was not carried out. This is often analyzed to compare different studies, to look which intervention is preferable. However, the confidence interval tells nothing about how the effect of study varies from study to study.

Prediction interval may serve as alternative, relative to the confidence interval.

When the prediction interval is greater than the confidence interval, the chosen intervention would not work out equally well. 't Hout's research shows that in 70% of the examined statistical significant meta-analyses the effect of the studied treatments was zero or even contrary to the average. In 20% of these metaanalyses this opposite effect was at least as big as the average effect of the metaanalysis (in 't Hout, 2015).



A power analysis was not carried out in this study. This should normally be carried out in order to be able to make a good estimation of the number of required

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observations to minimize type I and type II errors in the hypothesis testing period (Dolmans et al. 1994). Since there is no comparative research is was no option to the start before this this study a power analysis. By the numerous variables that were examined in this study a power analysis was not performed during the study because of the financial limitation. The sample was arbitrarily set at 40 subjects before the start of the study. An intention to treat analysis (ITT) was factored in advance, but was not analyzed for this study since the absence of withdrawals (Gupta, 2011). A power analysis as post-hoc test was not performed because of financial constraints.

Further post-hoc tests on ANCOVA can still be performed such as: the Sidak Pairwise Comparison and Bootstrap for Pairwise comparisons (Fiels, 2012). In further research, the results can be reanalyzed, for example in function of the cardiac autonomic balance versus the cardiac regulatory capacity (Brentson et al.

2008). Given the inter-and intra individual differences, a 'per individual analysis' in a group might be able to tell us more than the exported statistics. 

Bayesian statistics can provide an alternative to classical statistic because both the data (Likelihood function) as the unprecedented parameters (prior-distribution) and the data generating process are being approached stochastic. From the classical point of view only data is considered as stochastic and the data-generating process as deterministic (non-stochatic) (Wagenmakers et al. 2011; Gómez– Villegas et al. 2009).



Eventually, a Bonferroni correction on the p-values of the number of main hypotheses could be performed (Simmons et al. 2011).



Addendum 9 doesn’t mention all the results of this study. For reporting the statistical results the General guidelines of psychological research were took into account (At: http://evc-cit. Info/psych018/Reporting URStatistics. Pdf). The full results of this study can be obtained on request at the researcher, this to ensure the accuracy and objectivity (Simmons et al. 2011).

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10.4 Discussion techniques 

In literature there are various Osteopathic studies with the application of a single or multiple techniques (Henley et al. 2008; With, 2012; Milnes and Moran, 2007; Gfeller, 2012; Giles et al. 2013; Ruffini et al. 2015; Collard, 2009; Cantalino et al.

2014; Vander Mast, 2008; Girsberger et al. 2014).



On the basis of a 'black box' model scientific research can be carried out on the effect of an intervention that represents the practice situation (Van Tintelen, 2001).



The use of a set of techniques in a trial resembles more the practical situation in relation to the use of one technique. The disadvantage of applying multiple techniques is that no ruling out can be formulated to the effect of a single technique.



The terminology of manipulation is not always that clear. Manipulation, in the 'Glossary of osteopathic terminology', is described as follows "Therapeutic application of manual force". Indirect methods (techniques) are one of the thirtyseven possible subdivisions of an Osteopathic manipulative treatment (OMT). The definition of indirect techniques is described as follows "a manipulative technique where the restrictive barrier is disengaged and the dysfunctional body part is moved away from the restrictive barrier until tissue tension is equal in one or all planes and directions. Manipulation: therapeutic application of manual force" (Treffer et al. 2011). However, one can raise the question whether this division is relevant to research in practice.



There are inconsistencies in the existing literature on the therapeutic effect of cranial techniques (Cantalino et al. 2014; Mehl-Madrona et al. 2007; Green et al.

1999). Therefore it was deliberately chosen to apply only but a set of indirect cranial techniques to for this study. By using a mix of manual and visceral techniques, no ruling out can be formulated about the effect of a set of cranial techniques. Despite an integration of manual, visceral and cranial techniques resemble more the common practice situation.



The Sham consisted of ‘hands on techniques’ so that for the test subject it was hard to make out which group he or she belonged. The cure credibility can interact with the test subject his expectations and study design (Licciardone and Russo,

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2006). Research shows that only physical contact (hand-holding) has already an increasing effect on HRV (Pishbin et al. 2012), which we also determine in this research. 

There is still lack of common and rubust manual Sham (false treatment) paradigm.

It is forced to clarify and codify the Sham methods (used in manual therapy research) to asses and develop a robust paradigm that may become the standard Sham (placebo) arm of a trial (Cerritelli et al., 2016). Placebo interventions can improve physical (disease) processes of perpheral organs more easily and effectively than biochemical processes (Meissner et al. 2007). Expectations from the subject and/or administrator can influence the placebo effect (Gracely et al.

1985).

10.5 Discussion of the results Clinical trials show that there is a relationship between the ANS and the functioning of the body in state of health and a state of disease (Berntson et al. 1997; Thayer et al.

2010). Heart rate variability, skin conduction level, RSA, pre-ejection period and blood pressure are accepted parameters to formulate statements with regard to the ANS (von Borell et al. 2007; Bach et al. 2010; Joyner et al. 2010; Van Dijk et al. 2013; Sztajzel, 2004; Kleiger et al 2005; Benedek and Kaernbach, 2010; Sawyer et al. 2015).

Measuring the physiological reactivity of the autonomic nervous system can focus on two systems: the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenalMedullary (SAM) system. HRV, RSA, SCL, PEP and blood pressure can be used as an index for the SAM-activity and resemble the sympathetic and parasympathetic activity and arousal (Von Borell et al. 2007; Bach et al. 2010; Joyner et al. 2010; Van Dijk et al.

2013). HRV is analyzed in three domains (time domain, frequency domain and nonlinear analysis). HRV parameters in these domains: the total variability (Total power, SDNN, D2), parasympathetic activity (rMSSD, pNN50, HF ms2, HF n. U.), the influence of both branches of the ANS (average heart rate, IBI, respiration rate), the sympathovagal balance (LF ms2, LF n. U., LF/HF ratio), the scaling behavior of the ANS (DFA α1 en α2, SD1 en SD2) and the cardiac complexity (ApEn, ShanEntropy, D2) (Vandeput, 2010). LF/HF ratio is not an accurate reflection of the cardiac sympathovagal balance (Billman, 2013; Eckberg, 1997). When the heart rate is lower as 100 bpm the parasympathetic activity dominates, because of the intrinsic rate generated by the sinoatrial node. When the heart rate is higher than 100 bpm, there occurs a shift towards sympathetic activity (McCraty, 2015).

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Respiratory sinus arrhythmia (RSA) is an index for vagal cardiac control (van Dijk et al.

2013), while the pre-ejection period (PEP: the time between two cardiac events, the depolarization of the left vertices and the ejection of the blood in the aorta) resembles the sympathetic cardiac control (Licht et al. 2010; Norman et al. 2011). Skin conductance level (SCL) indicates the level of physiological arousal (arousal level) (Cacioppo et al. 2007; Doberenz et al. 2011; Benedek and Kaernbach, 2010; ).

Although PEP and SCL are exclusively sympathetically modulated, there is a fundamental anatomical difference in terms of innervation. Most postganglionic orthosympathetic fibers use norepinephrine as a neurotransmitter to the target organ (Îą-and β-receptors) (adrenergic innervation). The innervation of the eccrine sweat glands (SCL) is submitted by fibers from the sympathetic trunk, but doesnâ&#x20AC;&#x2122;t secrete norepinephrine but acetylcholine (muscarinic receptors) (orthosympathetic cholinergic innervation) (Shibasaki and Crandall, 2010; Bouman et al. 2008; Samuels et al. 2008 (a, b); Merckelbach et al. 1995; Wilke et al. 2009). It is being suggested that the reflection of sympathetic activity differs from the heart and skin (de Geus et al. 2014).

Blood pressure is subjected to both branches of the ANS (Bauer et al. 2002) and serves as only clinical effect size, while the other parameters act as intermediate effect size (De Cock, 2014). By assembling the above parameters nuanced statements can be obtained regarding the complexity of the ANS (van Dijk et al. 2013; De Geus and Van Doornen, 1996; De Geus et al. 1996).

Since the distribution knows a non-normal distribution, only at the basis of the average (arithmetic mean) incorrect statements can be made about the (treatment) effect on an increase/decrease of the variable. The average serves as center size and resembles the central tendency, but is sensitive to outliers (minimum-maximum). The median is less sensitive for these outliers but gives little information on itself (Out et al. 2014).

Twenty-eight parameters t1-t2 (experimental group, Sham) showed a contradiction between the mean and the median (Addendum 9). The mean and the median were taken into consideration to grant statements about the influence. Only for the variables that exhibited a (treatment) effect, the phenomenon of regression to the mean (RTM) was extra, per individual, investigated, which could be a cause of observed changes (Barnett et al 2005). RMT was not discussed in the analysis (Addendum 9).

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10.5.1

Discussion of the results : Comparison baseline (t0)

Experimental group with Sham Before the start of the experiment we could remark that on behalf of the baseline that the experimental population knew a higher overall variability, relative to the Sham group. Characteristic a suppression of the sympathovagal balance is noticed, with a domination of the parasympathetic branch of the ANS.

A reduction was found In the scaling behavior of the ANS for the short-term fluctuation (DFA α1) and long-term fluctuation (DFA α2), which’s strengthens the vagal dominance in the sympathovagal balance (Achary et al. 2006; Kleiger et al. 2005). An increasement of the short-term variability (SD2) implies an increased sympathetic activity in the sympathovagal balance (Hoshi et al. 2013), the contrary in the detrended fluctuation analysis (DFA). SD2 and DFA represent both the sympathovagal balance, but they demand a different analyzing method (Acharya et al. 2006; Muralikrishnan et al. 2013). From the Poincaré plot (Scatter plot), SD1 and SD2 were obtained by comparing the current R-R interval opposed to the previous R-R interval. SD2 (the length of the eclipse) represents the standard deviation of the continuous beat-to-beat R-R interval (long-term) variability (Muralikrishnan et al. 2013). Another method that was used is DFA to determine the fractal scaling properties with the long-distance correlations between the R-R intervals (Dutta et al. 2013). The experimental baseline showed an increase of the average heart rate with a decrease of HF n.u.(FFT, AR).

From this perspective, this implies an inhibition of parasympathetic activity on the cardiac vagal control, which contradicts the above.

The experimental baseline showed higher PEP values, which indicates on an inhibition of the sympathetic control of the heart (van Dijk et al. 2013; Brentson et al. 2008; Goedhart et al. 2008; van Lien et al. 2015). An increased baseline of the SCL indicates that there is a raise of sympathetic arousal (Cacioppo et al. 2007; Benedek and Kaernbach, 2010), while PEP showed an inhibition of the sympathetic activity in the cardiac control. From the activity from the electrodermal (skin conductivity level) it exhibits that the experimental population is in a higher physiological excitement (arousal level), relative to the Sham.

In the cardiac complexity was a sympathetic suppression noticed. A reduction of the complexity and regularity in the time series was observed (ApEn), which implies the lack of relative consistency (coherence) (Perakakis et al. 2010; Pinicus et al. 1991).

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Hereby it can be difficult to compare the different data sets with each other. A great distribution of the physiological patterns was present (ShanEntropy), which is featured in a flat distribution. The patterns were identically distributed, therefore the series contain a maximum amount of information.

The systolic and diastolic blood pressure was lower in the experimental baseline, which possibly may indicate an increased modulation of the parasympathetic branch of the ANS (rMSSD, pNN50, HF ms2) with a reduced activity of the baroreceptors and between the mix of the ANS (LFms2).

10.5.2

Discussion of the results: t1and t2 compared with

the baseline (t0) in the same group (Wilcoxon Ranks Test) and between the different groups (ANCOVA) 10.5.2.1

Total variance of HRV

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test), both interventions exhibit directly an increased influence on the Total variance of HRV. Indirect osteopathic cranial techniques and hands-on exhibited directly an increased significant influence on the global HRV index (SDNN), but could not be considered as an effect. An increase in the average Total power ms2 indicates an increasement of the physiological (emotional) regulation and adaptation (Thayer et al.

2012). Cranial techniques exhibited a higher degree on the sympathetic cardiac complexity and regularity of the physiological time series (ApEn) with a higher global autonomous variability (D2) (See infra: 9.2.1.2.6 Cardiac complexity and regularity).

On the short term, craniale techniques increases the Total variance of HRV, the opposite of the hands-on. A significant difference was found between the different groups (ANCOVA) in the short-term for: D2, SDNN. A trend was found for Total power ms2.

The applied techniques have no immediate or short-term (treatment) effect on: SDNN, Total power ms2, ApEn, D2 (H0 hypothesis could not be rejected). The Sham shows no immediate or short-term (treatment) effect for: SDNN, Total power ms2 , ApEn, D2. An upward trend for Total power ms2 [K] t2 (FFT/AR) was stood.

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10.5.2.2

Parasympathetic activity

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test), both interventions facilitate instantly parasympathetic activity. Hands-on exhibited directly a significantly increased effect on the instantaneous beat-to-beat (short-term) variability (SD1) and rMSSD, which implies an increased vagal modulation (Acharya et al. 2006; Singh and Bharti, 2015; Pumprla et al. 2002; Kleiger et al. 2005).

A direct increase of the cardiac vagal control (RSA) with a reduction in the average heart rate was felt in both groups, which induces an increase in parasympathetic activity (Moldovan et al. 2004; Barlow, 2007). Cranial techniques showed directly a reduction on the respiratory rate. Reduction of the respiratory rate is affected by parasympathetic activity, while an increase is modulated by sympathetic activity. In both groups, the heart rate was at least two times faster than the breathing frequency, which serves as a condition for the interpretation of RSA (Grossman and Taylor, 2007).

Both interventions exhibited in the frequency domain (FFT analysis) directly an increase in the cardiac vagal control (HF ms2), while the AR analysis demonstrated only a rise at the Sham. Software program VU-DAMS showed higher values of HF ms2 in opposition to Kubios. Hands-on exhibited a significantly reduced effect on HF n.u.

AR [K], the opposite of HF ms2. HF n.u.represents the relative value of each power component [(HF n. U.= HF/(Total power-VLF) x 100] and minimizes the effect of the absolute value of HF ms2. AR analysis gives relative lower HF n.u.values compared to the FFT analysis (Gernot, 2014; Sztajzel, 2004).

On the short-term, cranial techniques exerts an inhibitory role on the parasympathetic branch of the ANS (rMSSD, pNN50), while the hands-on elicits this. The same phenomenon on the vagal activity (supra) was seen for the instantaneous beat-to-beat RR-interval (short-term) variability (SD1) and at the cardiac vagal control (RSA). An increase on the respiratory frequency was observed in both groups, which implies sympathetic activity (de Geus et al. 1995). Cranial techniques showed on the shortterm an higher increasement of the average heart rate. On the contrary, the Sham presented, depending on the used HRV software program (VU-DAMS, Kubios), an increasement or decrease of the average heart rate ([V]: 1.70%, p >.05; [K]: -.14%, p >.05). The average heart rate for both groups was below the range of 100 bpm, which implies an inhibition/ excitation of the parasympathetic activity (McCraty, 2015; Bokhari et al. 2003). Give the contra version for the average heart rate in the Sham group makes it difficult to interpret RSA.

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Only on the short-term is significance found between the different groups (ANCOVA) for: rMSSD with a medium/large effect size, pNN50 with a large effect size, HF ms2 with a large/medium effect size, HF n.u. AR with a medium effect size, SD1 with a medium effect size, RSA with a medium effect size. A trend was found for HF n.u.FFT with a medium effect size.

The applied techniques only have on the short-term a lowering (treatment) effect on HF ms2 FFT [V] and HF n.u. AR [K] (H0 hypothesis is rejected). The applied techniques showed for HF n.u.FFT [K] t2 and RSA t2 none nor direct effect on the short term (treatment), but showed a downward trend. The applied techniques have no immediate or short-term (treatment) effect on: rMSSD, pNN50, SD1, Average HR, respiration rate (H0 hypothesis could not be rejected). Hands-on has no immediate or short-term (treatment) effect on: rMSSD, pNN50, SD1, Average HR, respiration rate, HFms 2, HF n.u.

10.5.2.3

Sympathetic activity

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test) the intervention elicits instantly sympathetic cardiac control (PEP), while handson performs an inhibition. Increased PEP value (long PEP value) are associated with an decrease in sympathetic cardiac control (Licht et al. 2010; Norman et al. 2011). Both interventions increases significantly the arousal level (SCL) or â&#x20AC;&#x2DC;energetic mobilizationâ&#x20AC;&#x2122; (Merkelbach et al. 1995), which implies an excitation of sympathetic activity (Cacioppo et al. 2007; Benedek and Kaernbach, 2010). Eccrine sweat glands are innervated sympathetic. The neurotransmitter in the post ganglion synapse of this system is acetylcholine and not noradrenaline (Wilke et al. 2009).

On the short-term, cranial techniques exhibited a decrease tendency of the sympathetic cardiac control, while the level of physiological arousal gives an increased trend.

Only directly is a tendency found between the different groups (ANCOVA) for PEP with a medium effect size.

The applied techniques have no immediate or short-term (treatment) effect on: PEP and SCL (H0 hypothesis could not be rejected). Hands-on has no immediate or shortterm (treatment) effect on: PEP and SCL.

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10.5.2.4

Sympathovagal balance

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test), both interventions exhibited directly an increase on LF ms2: the activity of the baroreceptors, between the mix of the ANS, the autonomic cardiovascular regulation system (Connes, 2010; Billman, 2013). Cranial techniques showed directly a reduction in the respiratory rate, while it increased by hands-on. A reduction in the average variance of LF ms2 can be made plausible because the reduction of the respiration rate is through the dominance of the parasympathetic branch of the ANS. An increase in the average variance of LF ms2 may be due to an increase in the respiration rate by the dominance of the sympathetic branch of the ANS. Both interventions showed directly an increased tendency in the autonomic cardiovascular regulation system (LF/HF ratio). LF/HF ratio has gained wide acceptance as a tool to assess cardiovascular autonomic regulation where increases in LF/HF ratio are assumed to reflect a shift to “sympathetic

dominance”

and

decreases

in

this

index

correspond

to

a

“parasympathetic dominance.” LF/HF ratio cannot accurately quantify cardiac “sympatho-vagal balance” either in health or disease (Billman, 2013; Connes, 2010).

On the short-term, cranial techniques exhibited an increase of the sympathovagal balance, while hands-on depending on the analysis (FFT: increase, AR: reduction) exhibited a ambiguous answer. An increase in respiration rate probably indicates an increased sympathetic regulation. In both analyses (FFT, AR) showed cranial techniques a significantly increased effect on LF n. U., which confirms the above.

Only on short-term, there is significance found with a medium effect size between the different groups (ANCOVA) for: LF n.u.AR, LF/HF ratio FFT [V]. Short-term trend with a medium effect size was also found for: LF n.u.FFT, LF/HF ratio FFT [K] and LF/HF ratio AR [K].

The applied techniques only have a short-term increased (treatment) effect on: LF n.u.

AR [K] (medium effect sizes) and LF/HF ratio FFT [V] (small effect size) (H0 hypothesis is rejected). A reduced tendency for the (treatment) effect was found for LF n.u.FFT [K] t2. The applied techniques don’t have an immediate nor short-term (treatment) effect on: LF ms2 FFT/ AR, LF n.u.FFT (H0 hypothesis could not be rejected). Hands-on has no immediate or short-term (treatment) effect on the above parameters.

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10.5.2.5

Scaling behavior of the ANS

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test) both interventions exhibited in the scaling behavior of the ANS directly an increase on the short-and long-term fluctuations (DFA: α1, α2) in the short-and longterm variability (SD: 1, 2). An increase of DFA (α1, α2) and SD2 indicates an excitation of sympathetic modulation in the sympathovagal balance (Acharya et al. 2006; Kleiger et al. 2005; Muralikrishnan et al. 2013). Both interventions exhibited directly a deceleration of the average heart rate and an increase in the average variance HF ms2.

From this perspective, this would imply an inhibition of the cardiac sympathetic control.

A direct increase of short-term variability is noticeable in both groups, which induces an increase of vagal activity (Singh and Bharti, 2015).

On the short-term, cranial techniques exhibited an increased trend in the short-term fluctuations (DFA α1), while this is lowers at the Sham. A significant increasing effect of the long-term fluctuations (DFA α2) is noticed at the cranial techniques, which implies an increased sympathovagal balance (Kleiger et al. 2005). The opposite is noticed in both interventions for short-term variability. Cranial techniques exhibited an inhibition on the vagus nerve, while this elicits by hands-on. The long-term variability (SD2) was reduced at the cranial techniques, while hands-on this significantly increased. A decrease of the long-term variability is associated with reduced sympathovagal activity (and vica versa) (Singh and Bharti, 2015). A decrease in the short-term variability (SD1) is being observed at the cranial techniques and is associated with reduced parasympathetic activity (Singh and Bharti, 2015), while this was increased for handson (excitation).

Only on short-term, there is significance between the different groups (ANCOVA) found for: SD1 with a large effect size, DFA α1 with a medium effect size, DFA α2 with a large effect size. Short-term trend with a medium effect size was also found for: SD2.

The applied techniques only have short-term increases (treatment effect) with a large effect size on: DFA α2 (H0 hypothesis could be rejected). A short-term trend was found for DFA α1. The applied techniques have no immediate or short-term (treatment) effect on: SD (1, 2), DFA α1 (H0 hypothesis could not be rejected). Hands-on has no immediate or short-term (treatment) effect to the above parameters, but a trend was found for SD2 on the short-term.

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10.5.2.6

Cardiac complexity and regularity

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test), cranial techniques exhibited directly an increase in the cardiac complexity and regularity of the physiological time series (ApEn), which was opposite for the hands-on.

An increase in ApEn indicates a relative consistency (coherence) by which the different time series can be compared and interpreted (Pincus et al. 1991). Both interventions exhibited a great distribution of the physiological patterns, characteristic with flat distribution (ShanEntropy). The patterns were distributed identically by which the series could offer a maximum amount of information (Kunz et al. 2012). Both interventions showed an immediate increased degree in the cardiac complexity of the ANS (D2), possibly by an impairment of sympathetic activity. A raise in D2 indicates an increased degree of freedom in the global autonomous variability (Bolea et al. 2014).

On the short-term, cranial techniques showed a significantly increased effect on the complexity and regularity of the physiological time series (ApEn). A difference in the distribution of physiological patterns (ShanEntropy) was present for the short-term between the experimental group (+) and Sham (-), but was not significant. A reduced degree of complexity in the ANS was observed for the cranial techniques, the opposite of hands-on. This might indicate on a suppression of the parasympathetic activity and an impairment of sympathetic activity (Pincus et al. 1991; Kunz et al. 2012). In the Sham probably other subsets of patterns are present, by which others are missing or hardly present. The patterns were not identically distributed, which made that the series carried only a minimal amount of information. A reduced degree of complexity in the ANS (D2) was found for the cranial techniques, the opposite of hands-on . On the short-term, this would indicate for the cranial techniques on an impairment of the parasympathetic activity, and a suppression of sympathetic activity for hands-on (Bolea et al. 2014).

Only on short-term, there is significance between the different groups (ANCOVA) found for: D2, with a large effect size.

The applied techniques have no immediate or short-term (treatment) effect on: ShanEntropy, ApEn and D2 (H0 hypothesis could not be rejected). Hands-on has no immediate or short-term (treatment) effect to the above parameters.

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10.5.2.7

Blood pressure

Compared to the baseline (based on the Mean) in the same group (Wilcoxon Ranks Test), cranial techniques exhibited directly an increase on the systolic and diastolic blood pressure, while this decreased at the hands-on. For the cranial techniques this would imply an excitation of the sympathetic activity, whether hands-on exhibited an inhibition.

On the short-term, hands-on exhibited a significant downward effect on the systolic and diastolic blood pressure. A decrease was also noticed for the cranial techniques.

In both groups is no immediate or short-term significance difference found between the different groups (ANCOVA) for: systolic and diastolic blood pressure. A trend was found for SBPL t1 with a medium effect size.

The applied techniques have no immediate or short-term (treatment) effect on the blood pressure. A trend was immediately found for SBPL (H0 hypothesis could not be rejected).

10.5.3

General

Different causes can affect the short-term changes in HRV: time changes, the course of changes in the sensitivity and specificity of the process, effect of the intervention (De Cock, 2016; Vermeulen, 2001). In complex systems surrogate parameters move in the direction to a point of transition. The direction in which the system makes a shift from one phase to the other phase (allostasis) takes place towards a possible threshold (tipping point). Each system contains a finite number of states of balance or quasibalance that can be achieved. Complex systems such as the human body are located in an attractor state, which is constantly disturbed by internal and external factors to be rebalanced (homeostasis). The direction of the system (and the velocity of changes) are more interesting as the state of the system itself (Scheffer et al. 2009). The phenomenon "Critical slowing down" was already observed in different systems (cellsignal catabolic pathways, ecosystems) and can lead to an increase in the autocorrelation in the fluctuating patterns (Wissel, 1984; Bagowski and Frell, 2001; Scheffer et al. 2009). The reduced modification is an equivalent of a point in the past, the system has a memory of itself. The phenomenon of autocorrelation can be measured by a frequency spectrum analysis (Livina and Lenton, 2007). The results in

136

this study show the importance of a follow-up to be included in a study (homeostatic versus allostatic).

137

11. Conclusion An experiment on the human person may only be performed if the study is approved by a (fully recognized) Medical Ethics Committee (CME). In addition, the study should have obtained a ‘definitive’ favorable opinion from a CME and completed the flawless civil liability insurance concerning experiments on the human person, which is a legal obligation. The RILHOU Randomized Clinical Trial (Eudract/B-nr: B371201422023) is performed in accordance with all the ethical and legal requirements/ obligations.

Results from this study demonstrated that both interventions (indirect osteopathic cranial techniques, hands-on) have an immediate increased influence on: total variance of HRV, parasympathetic activity, sympathetic activity on the level of physiological arousal, sympathovagal balance and scaling behavior of the ANS. Only the applied techniques have an immediate excitatory influence on: sympathetic cardiac control, complexity and regularity of the physiological time series and the systolic/diastolic blood pressure. On the short-term, only the applied techniques have an inhibitory

influence on the total variance of HRV and the parasympathetic activity, while sympathovagal balance, scaling behavior of the ANS and sympathetic cardiac complexity is encouraged. Both interventions exhibited an inhibition on the sympathetic cardiac control and on the systolic/diastolic blood pressure, while the level of physiological arousal increases.

In the examined sample population the applied techniques only exhibited a short-term (treatment) effect on HRV (frequency domain: LF/HF ratio FFT [V], LF n.u. AR [K], HF n.u. AR [K], HF ms2 FFT [V], nonlinear analysis: DFA α2. The applied techniques showed no immediate and short-term (treatment) effect for the other HRV parameters, SCL, RSA, PEP and blood pressure. Adverse events did not occur in this study.

Further research with a separate control group and stratification (gender, age, respiration rate) is necessary to formulate further statements. The study focused on healthy subjects. Future research is needed to explore the extent to which indirect osteopathic cranial techniques can change ANS activity in pathological conditions and to compare this effect with standard healthcare, hypothesizing that indirect osteopathic cranial techniques could be used as a supportive care in addition to traditional methods.

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13. List of abbreviations ACE:

Angiotensine I Converting Enzyme

AD:

analoge-digitale

AMD:

Ambulatory Measurement Device

ANP or ANF:

Atrial Natriuretic Peptide

ANS:

autonomic nervous system

ApEn:

Approximate Entropy

AR:

Autoregressie

ASDNN (index):

Average of standard deviation of all NN intervals

B. S.:

Belgian Official Journal

BAKO:

Belgian Association of Classic Osteopaths

BMI:

Body Mass Index

bmp:

beats per minute

BNP:

Brain Natriuretic Peptide

BP:

blood pressure

BUO:

Belgian Union of Osteopaths

BUPO:

International Covenant on Civil and political rights

BVO:

Belgian society for Osteopathy

CAM:

Complementary Alternative Medicine

CER:

Clinical Effectiveness Research

CI:

Cardiac index

CME:

Committee on Medical Ethics

CNP:

C-type Natriuretic Peptide

CO:

Cardiac output

CORPP:

Commission for Osteopathic Research Practice and Promotion

CROaM:

Clinical Risk Osteopathy and Management

D2:

Correlation Dimension Analysis

DBPL:

diastolic blood pressure right

DBPR:

diastolic blood pressure left

DFA:

detrended fluctuation analysis

DO:

Doctor of Osteopathy (USA)

D. O.:

Diploma in Osteopathy (Europe)

Dr.:

Doctor

DTS:

detrended time series

dZ/dt:

impedance changes over time

ECG:

electrocardiogram

184

EDA:

electrodermale activity

EDR:

ECG derived respiration

EEG:

electroencephalogram

EG:

Directive European Union

EU:

European Union

EU-GCP:

Europese Good Clinical Practice

EVRM:

European Convention on human rights

Expi.:

Experiment

F. I. C. O.:

Flanders International College of Osteopathy

FDA:

Food and Drugs Administration

FFT:

Fast Fourier Transform

FMEA:

Failure Mode and Effects Analysis

GCP:

Good Clinical Practice

GMP:

Good Manufacturing Practice

HF:

high frequency power

HR:

heart rate

HRV:

Heart Rate Variability

Hz:

Hertz

IBI:

interbeat interval

ICF:

Informed Consent Form

ICG:

impedance cardiogram

ICH-GCP:

International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use. Good Clinical Practice

ITT

Intention to treat analyse

K. B.:

Royal Decree

KHz:

Kilohertz

LF/HF:

ratio of the low-high frequency power

LF:

low frequency power

LVET:

left ventricular ejection time

mm:

millimeters

ms:

milliseconds

2

ms :

square milliseconds

MSc:

Master of Science

n. U.:

normal units

NCOR:

National Council for Osteopathic Research

185

NN50:

the number of pairs of successive NNs that differ by more than 50 ms

Nr.:

number

PDS:

power spectral density analyse

PEP:

Pre-Ejection Period

PhD:

Doctor of Philosophy

pNN50:

the proportion of NN50 divided by total number of NNs

R. O. B.:

Register of Osteopaths of Belgium

RCT:

Randomized Clinical trail

RILHOU:

Acronym of the second letter of each researcher his/her name.

rMSSD:

root mean square of successive differences

RRtri:

Triangular index

RSA:

Respiratory Sinus Arrhythmia

SampEn:

Sample Entropy

SBPL:

systolic blood pressure left

SBPR:

systolic blood perssure right

SC:

skin conductance

SCL:

Skin Conduction Level

SCR:

skin conductance response

SD HR:

standard deviatie of all heartbeats

SDANN:

standaard deviatie of the average NN intervals

SDNN:

standard deviation of NN intervals

SNA:

sympathetic nervous activity

SV:

stroke volume

SVR:

systemic vascular resistance

TINN:

triangular interpolation of RR intervals

U. K. O.:

Union of graduate in the physiotherapy and osteopathy

U. V. O.:

Union Of Osteopaths

UK:

United Kingdom

ULF:

ultra low frequency

USA:

United States of America

VZW:

vereniging zonder winstoogmerk

WHO:

World Health Organisation

WMA:

World Medical Association

ZOL:

Ziekenhuis East-Limburg

[K]:

Kubios

186

[V]:

VU-DAMS

ηp2:

parial eta squared

Z0:

Thoracic impedance

µA:

microampère

μS:

microsiemens

187

14. Addenda

188

14.1

Addendum 1: Informed (translated dutch version)

consent

(De

Cock,

2014)

INFORMATION FORM (‘Informed Consent’ Form ) As a participant in this research we'll give you honest, objective information. You can completely run through this document and pose your questions. Only when everything is clear and you wish to participate in this research, we will ask you to date and sign the ‘Consent form' for agreement.

Title of the clinical experiment Effects of indirect Osteopathic techniques on 1) the heart rate variability including nonlinear analyses- and 2) electrodermal activity in healthy adult (obese) ¹. The RILHOU ² randomized clinical trial.

Effects of indirect osteopathic techniques on heart rate variability -nonlinear analyses included- and on electrodermal activity in healthy (obese)¹ adults. The RILHOU² Randomized Clinical Trial. ¹ In one partial research there are subjects with a BMI > 30. ² Acronym: the second letter of the first name of the participating researchers For General info Study coordination: VZW Health, Klein Lafeltstraat 21, 3770 Riemst, Belgium under the supervision of Smeets Hubert, DO. Phone: +32(0)124 5 6148 For specific questions A list of the cooperating researchers is always available at your disposal. Contact details of the researcher whom you will end up, will be given when you’re in treatment.

Background of the study The human heart rate has no fixed rhythm, but hovers around an average heart rate frequency. At one time, your heart beats 70 times a minute, other times a bit faster, for example 80 times per minute. On an electrocardiogram (ECG) one registers this heart rate activity.

An ECG should then look like this:

189

Every time you feel your heart beat, this corresponds to the pike on the ECG, the Rtop. If your heart beats fast, one heart-beat will follow faster on a previous one. Hence the distance between the R-tops reduces. When your heart beats slower, the distance between the R-tops will increase.

The time lap between 2 R-tops on an ECG is called the R-R interval. On a normal ECG one speaks also of a NN interval (Normal to Normal).

The different variations in the R-R interval on a normal ECG are a normal physiological phenomenon. From of the R-R intervals, the heart rate variability (HRV) is extracted. A high heart rate variability is associated with good health. A low heart rate variability is associated with various diseases.

Sweat glands in the skin are stimulated to secrete sweat through some of our nervous system (sympathetic nervous system). According to the physical effort and also by certain emotions -think of fear sweat- more or less sweat is secreted. Many substances -also our skin- conducts more or less electricity. When the sweat secretion changes, also the electrical conductivity of the skin will change. Electric skin activity is one of the most measured physiological signals.

By using a device (the VU-AMS), we wish to register both the heart rate variability and the skin conductance level. These data can be further analyzed using customized software.

Importance of our research Type and purpose of the study/the experiment Scientific research within osteopathy is highly recommended by the WHO. The influence of Osteopathic techniques on the heart rate variability is already being examined a few times. However, there is a lack of good research according to a strict methodology and with a sufficient number of subjects.

To achieve our diploma in osteopathy (a DO or a ' Master '), we are required to have a thesis that we need to defend in front of a jury. The thesis should cover carried out research.

This is the reason why we want to examine the effect of various indirect Osteopathic techniques on the heart rate variability (HRV) and on the skin conductance level with adults.

Brief summary of the project This trial will be carried out on 1 location: Flanders International College of Osteopathy, santvoortbeeklaan 23, 2100 Deurne, Belgium The total examination consists of a few partial investigations. Therefore we need healthy Dutch speaking persons, both women and men between 20 and 50 years. The Body Mass Index must be less than 30, but for one partial research we are also looking for people with a BMI > 30.

If you would like to contribute to our research, we will ask you a few questions and we will measure your weight and your height. If you are eligible for our research, then an appointment for treatment is made. All applied techniques are all very soft techniques.

190

Nature of the techniques During our training as a student, we have learned to practice these technique(s). We apply them on a regularly basis in our office and have them prepared extra for this trial.

They are described in the literature. During the study, no manipulations will be performed. All techniques are very soft techniques!

Risks and inconveniences and measures related here So far, there are no risks and/or side effects known or described for the applied techniques, but these can not be excluded. If side effects would occur, these will be approached and followed with the best care and ability, Before the start of the study your family doctor will be contacted regarding participation in this trial. The contact details of your family doctor will be included in your file.

During the experiment you are under constant supervision and this can be further assured during the following hours. During the investigation nursing permanence is foreseen. During the investigation and after researching, you have the option to appeal for: 1) the researcher , 2) your family doctor, 3) the Coordinator of the research.

If necessary, the on-call doctor can be consulted.

The contact details of this doctor will also be included in your file.

Implications participant: what is expected from you in this study?

We would like to ask you some questions and register your height and weight. If you are eligible for participation, you should be able to move twice to Deurne. The first time, a pair of electrodes with a gel will be set at certain places to register an ECG and skin conductance. You have to lay down quietly in a supine position for approximately 15 minutes. Then you will get a soft treatment on the head and/or on the body and after that treatment the device remains registering further for a couple of minutes while you will remain in supine position. The total duration of the first session is limited to a maximum of 60 minutes. The second time, 7-10 days after the previous measurement/treatment, there will only be one measurement. The electrodes will be attached and the device will register while you are quietly lying down..

Voluntary nature of participation Participation in the study is completely voluntary and non-binding. You are qualified, at any time, to quit the participation in this trial without giving any reasons. Participation can also be cancelled by the researcher for a thorough, motivated reason in case of non-compliance. In this case, a written report will be attached to your file.

Benefits for the participant There cannot be guaranteed that you will benefit any direct advantage by participating in this trial. Based on the practical experiences of the researchers, it is hold that the applied Osteopathic techniques may have a positive effect on the HRV. Research has shown that a positive impact on HRV by means of the quoted therapeutic techniques is plausible.

191

Pressure on the participant No physical nor psychological effort is expected from you.

Responsibility for the confidentiality of the data Besides the medical information, also the data that are collected for the purposes of this study are extremely confidential. This complies with the medical secret, the international guidelines (ICH-GCP) and the Belgian legislation (including the legal requirements as set out in the Belgian law of 8 December 1992 on protection of privacy and the Belgian law of 22 August 2002 on the rights of the patient). If you wish to discontinue your participation in the study, no further new data will be collected. The data already collected can be used for analysis.

Encoding of the study data: The study data are encoded before they are transferred to a third party, i. E. entitled persons or institutions will only receive the data only after encoding. The randomization code (link between you as a participant and your data, in order the anonymize your data) is stored by the Coordinator of the research. Therefore, he is the only person who can associate code with participant.

Hence, the collected data will contain no elements of which the combination could lead to your identification as a participant by third parties (e. G. combination of initials, sex and full date of birth).

Direct access to the medical records by third parties: Under strict conditions direct access can be granted to the investigation file and/or medical file to authorized staff of official authorities, to regulatory authorities or to other persons who are authorized by law to ensure the trial is properly implemented. Access is given only to control the quality of the collected data, and is not systematically granted. Direct access to the medical records by third parties will only be granted in the presence and under the responsibility of the Coordinator (or of one of his employees).

All persons who may have access to the medical records are held by professional secrecy.

Rights of the participant: You as a participant always have the right to ask the researcher which information about you is being collected in the framework of the study and what its purpose is. You also have the right to access your personal information and, if necessary, to apply the necessary improvements.

Treatment after the study You always have the right, after the study to acquire the techniques used in this research.

192

Costs/financing Participation in the study will have no costs for you nor for your health insurance.

Statisticians, supervisors and other employees will be able to be reimbursed in the context of potential funds and/or sponsorship and this in consultation with the persons concerned. Currently there are neither funds nor sponsorship.

Insurance In accordance with the Belgian law of 7 May 2004 on experiments on the human person, the sponsor (initiator of the study) can be held liable for any damages to the participant or his/her assignees, which are directly or indirectly related to the experiment. The ordering party [sponsor] of this study has an insurance policy that covers liability. In case there would occur any damage to you as a result of your participation in this study, this damage will be covered in accordance with the Belgian law of 7 May 2004.

Approval by Central and/or local medical ethics Committee â&#x20AC;&#x153;This study was approved by an independent Ethics Committee (Committee on medical ethics in Genk, which acts as a Central Commission for this project). This study shall be carried out in accordance with the guidelines for good clinical practice (ICH/GCP) and according to the most recent version of the Helsinki declaration drawn up to protect people participating in clinical trials. In no case, the approval by the Commission for medical ethics must be considered as an incentive to participate in this trial".

193

14.2 Addendum 2: letter of agreement and consent form (De Cock JJ, 2014a) LETTER OF AGREEMENT VOLUNTARY PARTICIPANT Researcher (name)

AND

CONSENT

FORM

OF

THE

Code:

Effects of indirect osteopathic techniques on heart rate variability nonlinear analyses included- and on electrodermal activity in healthy (obese)* adults. The RILHOU² Randomized Clinical Trial. (* If applicable).

I declare that I am legal capable, that I have the legal capacity and voluntarily participate in this study.

I have received a full explanation about the background and purpose of the study team, the purpose and the expected duration of the experiment, the voluntary nature of the participation as well as that it is not guaranteed that I, as a participant, will personally benefit any direct advantage from participating in this study.

It is possible that soft indirect Osteopathic techniques will be applied on the head and on the body. In the common literature there exist no risks and/or side effects for these techniques, they can be considered safe. Also in practice no risks/side effects for these techniques are known. However, this cannot be excluded. Therefore, the researcher has, before the start of the experiment, notified my family doctor about my participation in this study.

I am also informed that during the whole experiment an electrocardiogram (ECG) and skin conductance is registered through a device, the VU-AMS. All the obtained data can be post processed with specialized software while maintaining my integrity and anonymity as a participant as described and explained under the heading ' responsibility for the confidentiality of the data '.

I received a clear insight on the ' Informed consent ' form and a copy was made available to me relating to this study. This form went through completely together with the researcher before this ' letter of agreement and consent form of the voluntary participant ' was signed.

I had the opportunity to ask the responsible researcher questions about his identity, accessibility and all aspects of the study. The information was provided in understandable language and was understood by me.

I have the opportunity to consult with family/acquaintances.

I have been informed that this is a one-time session with no heavy exposure. After 7 to 10 days a second measurement (follow-up) will take place. I am aware that there is no financial compensation related participating in this trial. Both for me -as a participantas for my health insurance it will not involve additional costs.

194

I fully understand that I am free to leave the study at any time without having to justify my decision and without further consequences. I’m aware that I can contact the Coordinator of the research after a possible termination of my participation in the study.

Although my name does not need to be provided in the study report that is transferred to a third party, I expressly give the researchers the consent to pass the results of this study to competent medical authorities (in Belgium or abroad). The data emerging from the trail, can be used at a later date for publication.

For agreement, the voluntary test subject, Name and surname:.....................................

Birth date......./......./....... To Deurne on...../....../.....

(Handwritten: for read and approved) Signature:

____________________________________________________________________ I, the researcher or his/her representative, confirm that I have seen the person mentioned below: name, surname:.......................................

I confirm that I gave him/her the necessary information about this trial and that I, in his or her presence, went through together the ‘Informed Consent Form’ and that I didn’t exercise any pressure in order that he/she would take part in this study.

I confirm having provided an auto-signed’ letter of agreement and consent form’ and that I am willing to answer all possible additional questions. .

I confirm that I work in accordance with the ethical principles as stated in the "Declaration of Helsinki" and the Belgian law of 7 May 2004 on experiments on the human person.

Signature ( name and first name)

To Deurne on...../....../........

195

14.3 Addendum 3: Flowchart of the four phases at The RILHOURCT Table 11: Flowchart of four phases of a parallel randomized clinical trial, modified from the Consolidated Standards of Reporting (CONSORT) (Schulz et al. 2010)

Assessed for eligibility (N= 56) By means of a randomized questionnaire Excluded (N= 16): Not meeting inclusion criteria (N= 6) Declined to participate (N= 2) Other reasons (N= 8) Randomised (N= 40)

Allocated to experiment (N= 20):

Allocated to Sham (N= 20):

Received allocated experiment

Received allocated Sham (N=

(N= 20)

20)

Lost to follow-up (N= 0) Discontinued experiment (N= 0)

Analysed (N= 20): Excluded from analysis (N= 0)

Lost to follow-up (N= 0) Discontinued Sham (N= 0)

Analysed (N= 20): Excluded from analysis (N= 0)

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14.4 Addendum 4: Endocrine heart Table 12: Substances produced by the vertebrate heart (Tota et al. 2010; with personal additions) Substance

Site of production

Reference

Adenylpurines

Endothelial cells and cardiac myocytes Shah, 1996

Adrenomedullin

Cardiac myocytes and fibroblasts (atria Kato et al. 2003 and ventricles)

Aldosterone

Atria and ventricles(rat)

Delcayre et al. 2000

Angiotensin II

Cardiac and vascular RAS

Dzau, 1993; Shimano et al. 2012

Atrial natriuretic peptide (ANP)

Cardiomyocytes

Clerico et al. 2011; Ogawa et al. 2014; Fukuma et al. 1999; Pandey, 2008

Apelin

Neonatal heart

Brain Natriuretic Peptide (BNP)

Cardiomyocytes

Katugampolaet et al.

2001 Clerico et al. 2011; Ogawa et al. 2014; Fukuma et al. 1999; Pandey, 2008

Calcitonin generelated peptide

Atria, sinoatrial and atrioventricular nodes

Beaulieu et al. 1998

Catecholamines

Myoendocrine, vascular and endocardial endothelial

Burnstock, 1969; Abrahamsson et al.

1979; Ebert et al. 2001; Ebert et al. 2006

Presumptive SA node

Cells, intracardiac chromaffin cells, ICA Larsen et al. 1994; Huang et al. 1996; Intrinsic cardiac adrenergic cells

Huang et al. 2005; Saetersdal et al. 1975

Chromogranin A and derived peptides

Atrial and ventricular cardiocytes, conduction system

Coenzyme A glutathione disulfide (CoASSG)

Myocardial tissue

Steiner et al. 1999; Weiergraber et al.

2000; Pieroni et al.

2007 Luo et al. 2006

C-type natriuretic peptide (CNP)

Endothelial cells and cardiac fibers

Clerico et al. 2011; Ogawa et al. 2014

Cytokines

Cardiac myocytes and coronary endothelium

Kaye et al. 1996

197

Endothelin-1

Coronary endothelium and cardiac tissue

Shah, 1996; Beaulieu et al. 1998

Ghrelin

Cardiomyocytes

Iglesiaset et al. 2004; Kishimoto et al. 2012

Growth defferentiation factor (GDF)-15 Interleukin (IL)-23

cardiomyocytes

Shimano et al. 2012

cardiac fibroblast

Shimano et al. 2012

Leptin

Cardiomyocytes

Purdham et al. 2004

MED13

Cardiomyocytes

Nakamura et al. 2014; Grueter et al. 2012

Migration inhibitory cardiomyocytes factor (MIF) Myostatin cardiomyocytes Neuropeptide Y

Shimano et al. 2012 Shimano et al. 2012

Nitric oxide

Nodal tissue, atria and coronary Beaulieu et al. 1998 vessels cardiac endothelial cels and vascular Shimano et al. 2012 cells Endothelial cells and cardiac myocytes Shah, 1996

Oxytocin

Cardiac tissue (rat)

Neuregulin

Jankowski et al. 1998

Proadrenomedullin Cardiac myocytes and fibroblasts (atria Kato et al. 2003 N-terminal 20 and ventricles) peptide (PAMP) Prostanoids

Endothelial cells and cardiac myocytes Shah, 1996

Protease inhibitor 16 (PI16)

Cardiomyocytes

Shimano et al. 2012

Relaxin

Atrial cardiomyocytes(rat)

Taylor and Clark, 1994

Substance P

Sinoatrial and atrioventricular nodes

Beaulieu et al. 1998

Urocortin 1 and 3

Cardiomyocytes and noncardiomyocytes

Ikeda et al. 2002; Takahashi, 2012

Urodilatin

Cardiomyocytes

Clerico et al. 2011; Ogawa et al. 2014

Vasoactive intestinal peptide (VIP)

Sinoatrial node and coronary vessels

Beaulieu et al.1998

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14.5 Addendum 5: The influence of cardiac hormones on HRV Table 13: The influence of cardiac hormones on HRV Substancesproduced by the vertebrate heart Adenyl purines

Results

Conclusion

Reference

SDNN was strikingly greater in type 5 adenyl cyclase knock-out Total autonomic variability index Okumura et al. 2008 (AC5KO) under micro (~40-fold)- and hypergravity (~9-fold).

(SDNN) was increased to a greater degree in AC5KO.

Adrenomedullin (ADM); Brain LF/HF and the concentrations of plasma ADM were significantly natriuretic peptide (BNP) reduced at 3 (rd) day and 3 (rd) mounth after transcatheter closure (P <0.01). The HRV spectral values (TF, VLF, HF) were significantly increased. LF/HF and the concentrations of plasma BNP were significantly higher in patients with PDA before.

LF/HF and plasma BNP were significantly decreased at 3 (rd) month after transcatheter closure (P< 0.001 or P <0.05).

HRV and plasma ADM, BNP improved Wu et al. 2010 significantly post transcatheter closure in childeren with patent ductus arteriosus (PDA).

Angiotensin II ; Aldesterone

The HRV index had good correlation with part of RAAS index.

SDNN, rMSSD, pNN50 were negative correlated with plasma angiotensin II (r= -0.438; P < 0.01; r= -0.592; P < 0.001; r= 0.786; P < 0.01) Respectivley SDNN, rMSSD and pNN50 had no obvious correlation with plasma aldosterone.

The HRV index such as SDNN, Mu, 2009 rMSSD and pNN50 all decreased in patients with paroxysmal supraventricular tachycardia (PSVT).

The level of plasma angiotensin II and aldosterone increased. There is a good correlation with plasma level of angiotensin II.

Apelin

Plasma apelin concentration of paroxysmal atrial fibrillation The down regulation of apelin China papers, 2010 patients was inversely correlated with the rMSSD (P= -0.386; concentration may related to the onset P= < 0.05) and pNN50 (P= -0.369; P < 0.05). of paroxysmal atrial fibrillation induced by hegh tension from the vagus nerve.

Atrial natriuretic peptide (ANP) Negative correlation between plasma ANP and the LF/HF ratio.

A close relationship between ANP and Kasamaki et al. 2013 cardiac parasympathetic and/or sympathetic activity.

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Brain (BNP)

natriuretic

peptide The highest Nt-proBNP quartile group had the lowest SDNN, The importance of Nt-proBNP levels to Lorgis et al. 2012 LF/HF and total power but similar pNN50 and rMSSD levels. Nt- predict decreased HRV after acute proBNPlevels correlated negatively with SDNN (r= 20.29; P= myocardial infarction (MI).

0,001) but not HF (r= 20.043; P= 0.172). Multiple regression analysis showed that plasma propeptide levels remained predictive of LF/HF (B(SE)= 20.065 (0.015); P= 0.001).

Calcitonin generelated CGRP concentrations were significantly lower in coronary heart peptide (CGRP); Substance P disease (CHD) group than that in the controle group (P < 0.05).

(SP) SP levels in plasma were lower in CHD group as to the control group (P= < 0.01). SSNN index in CHD group was significantly lower than the controle group (P= 0.01).

Catecholamines

CGRP and SP with lower level, HRV ShenSheng, 2011 reduced the parameters were closely related with the degree of coronary artery stenosis.

Low frequency was correlated with norepinephrine (r = 0.68; P= There is a strong correlation between Sesay, 2008 < 0.001). low frequency HRV, plasma norepinepherine, arterial blood pressure and heart rate during pheochromacytoma surgery.

Chromogranin A (Chga) and Catestatin (CST) increased the LF/HF ratio only in KO mice. derived peptides SDNN values were significantly lower in KO mice (P= < 0.003), which was reversed by CST (P= 0.0009) and significant reductions in S1 (P= < 0.05) and S2 (P= < 0.005) in KO mice.

Chga has a profound effect on Dev, 2010 autonomic tone to the heart and that its CST fragment is responsible for such actions.

Cytokines

There is a significant correlation between cytokine determination and several basline HRV parameters (pNN50: r= 0.42, P < 0.05; HF r= 0.4, P= 0.05; LF/HF: r= -0.43, P= < 0.05) on TNF alpha release following endotoxin.

There is a correlation between Jan et al. 2010 parasympathetic/ vagal parameters of HRV (HF and pNN50) and maximal TNF alpha level but not for IL-6 response following endotoxin exposure.

Endothelin-1 (ET-1)

The low heart rate variability group showed significantly lower Plasma ET-1 level was associated with Lee et al. 2012 ET-1 levels compared with the high heart rate variability group HRV parameters in patients with (0.57 ± 1.09 versus 2.21 ± 3.56 pg/ml, respectively; p = 0.04). normal-tension glaucoma.

Among the HRV parameters, the SDNN and Total power were significantly associated with the systemic level of ET-1 (P= 0.729, P= < 0.001; P= 0.713, P= < 0.001, respectively).

Gherlin

Gherlin decreased significantly the LF and LF/HF ratio of HRV Gherlin elicited a marked increase in Soeki et al. 2014 and increased the SDNN, rMSSD, and HF. circulating growth hormone, but not insulin-like growth factor-1.

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Leptin

Neuropeptide Y (NPY)

Body mass index (BMI) and percent body fat significantly modified the association between leptin and LF (but not HF) HRV. Among officers with BMI < 25 kg/m(2), leptin was not significantly associated with HRV. However, among officers with â&#x2030;Ľâ&#x20AC;&#x2030;25 kg/m(2).

Leptin levels were inversely associated Charles et al. 2014 with LF HRV, especially among officers with increased adiposity.

Increased leptin levels may be associated with CVD-related health problems.

Compared with the systemic sclerosis (SSc) and healthy Autonomic dysfunction and NPY are Di et al. 2009 controls (NHS) groups, the fibromyalgia (FM) group had curcial elements in the pathopysiology significantly higher NPY levels. The FM subgroup subjected to of FM. HRV analysis, (25/51 patients, 49%), certain HRV indices were significantly correlated with the SDNN index and the NN50, but neither NPY of HRV parameters showed any significant correlation with clinical aspects of the FM

Nitric oxide (Nox); Endothelin- Nox levels in group A (coronary slow) were lower than the 1 (ET -1) control group (23.4 +/- 13.5 micromol/L versus 42.5 +/- 15.9 micromol/L, P= < 0.05). Timed domain HRV parameters were prominent decreased in group A. TIMI frame count was also significantly correlated with ET-1 and Nox levels (r= 0.61, P= <0.0001; r= -0.30, P= < 0.05).

Patients with CSF, both increased Pepdemir et al. 2004 plasma ET-1, decreased plasma Nox and diffuse atherosclerosis may cause the decrease in HRV be effecting myocardial blood flow.

Oxytocin

Acute administration of OT may Kemp et al. 2012 facilitate a fundamental psychophysiological feature of social behaviour, increasing capacity for social engagement. HRV changes may provide a novel biomarker of response to OT nasal spray that can be incorperated into research on response to treatment.

HRV was increased under OT relative to placebo conditions for both detrended fluctuation scaling exponent (DFA alpha 1) [t(16)= 2.13, P= 0.02; d=0.47] and HF [t(16)= 1.468, P= 0.008; d= 0.27].

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14.6 Addendum 6: variables that have an impact on various parameters.

14.6.1 Relationship age, gender and HRV HRV values are higher in physical young, active and old women (Davy et al. 1996; Ryan et al. 1994). Newborn girls have a higher heart rate variability, compared to boys (Nagy et al.

2000). Bonnemeier et al. (2003) studied the heart rhythm variation of healthy persons between 20 and 70 years old. Their findings include that HRV reduces with age, more with women than men. Previous studies show that HRV related differences exist between gender and age in: time domain, frequency domain and some non-linear HRV analysis (Ramaekers et al. 1998). Age has a greater impact on HRV than sex. The total power HRV, LF and HF gradually decreases with age. Gender has no effect on SDNN and total power HRV (Zhang, 2007).

14.6.2 Relation gender and HR Gender would have a significant effect on heart rate (Zhang, 2007). A positive correlation was found in men between heart rate and age (r = 0.367, p < 0.01), with an increase in the sympathetic activity while aging. In women no correlation was found between age and heart rate (r= -.086, p .620). Only a minimal increase in heart rate was observed between the age groups (Yukishita et al. 2010).

14.6.3 Relationship age and SCL According while getting older, the skin will become drier compared to young age.

Electrodermal activity decreases with age (Doberenz et al. 2011).

14.6.4 Relationship sex and SCL With women, the electrodermal activity is affected by monthly hormonal variations (Goldstein et al 2005). Some studies confirm no difference between age and gender in SCL and SCR (Carrillo et al. 2001; Gavazzeni et al., 2008), while other studies showed higher SCL levels for men compared with women (Kronholm et al. 1993; Kelly et al. 2006). Some of these differences disappear depending on the age (Doberenz et al. 2011; Eisdorfer et al. 1980).

14.6.5 Status and HRV Donoho et al. (2015) examined the relationship between 'married, divorced, never married and widows' in relation to HF (HRV). No significant differences were found for HF between married couples, divorced, never-married and widows. Married individuals showed higher HF values compared to unmarried ones. Increases in marital satisfaction with support over more than 10 years were associated with higher HF values. An increase of marital stress over more than 10 years was associated with lower HF values.

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14.6.6 Status and BD Lipowicz and Lopuszanska (2005) examined the systolic and diastolic blood pressure of 2.271 healthy men between 25 and 60 years in relationship with their civil status. This study showed that never married men have (based on the mean) higher systolic and diastolic blood pressure, compared to married men.

14.6.7 Status and BMI Men who never have been married showed lower BMI values compared to married men (Lipowicz and Lopuszanska, 2005).

14.6.8 Relationship fear and HRV Major depressive disorders and anxiety are significantly associated with a lower HRV. Both associations are mainly caused by the effects of antidepressants (Licht and Penninx, 2009).

When experiencing safety or when a former-threatening signal is absent, the HRV reflects at rest an inhibition on the subcortical fear response (Wendt et al. 2015; Pappens et al. 2014; Thayer et al. 2012).

14.6.9 Relationship autoimmune disease and HRV Supine patients with celiac disease have a significant decrease in LF n.u.and HRV index (Przybylska-Felus et al. 2014). Compared to a healthy population a reduction for SDNN was determined with rheumatoid arthritis while pNN50 and rMSSD exhibited an increase (p >.

05). A reduction was seen in the frequency domain for HF with an increase of LF (p >. 05). A significant increase was found for LF/HF (Evrengül et al. 2004). Patients with Sjögren's syndrome showed a slower heart rate, larger R-R variability and higher SDNN values, compared with a healthy population. They found a small reduction of LF with an increase of HF. These differences were not significant. Significance was only found for a decrease of LF/HF ratio (Tumiati et al. 2000).

14.6.10 Smoking and HRV Relationship At nighttime, within 5 minutes after smoking a cigarette a significant increase of LF/HF appears. During the day this was not found. The increased reactivity of LF/HF and HF was more prominent present during night compared to the day. At night, smoking has more influence on the acute effect of the cardiac modulation compared to daytime. (Kobayashi et al. 2005).

Karakay et al. (2007) compared the HRV parameters with 5 minutes before and after smoking, etc. until 30 minutes after smoking. Compared with the baseline for the first 5 minutes after smoking, a significant reduction was found for the average R-R intervals, SDNN and rMSSD, while after a period of 20-30 minutes SDNN increased. In the first 5 minutes after smoking a significant reduction in LF/HF was observed. This reduction remained unchanged during the entire study period. An increase of LF and HF power was noticed after the first 5 minutes after smoking a cigarette. The abstract didn’t mention if the research took place during day or at night. In case of a non-smoking period, pNN50 and HF increase significantly, this both during day and night (Minami et al. 1999).

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14.6.11 Impact of smoking on blood pressure and heart rate During a 24-hour ambulatory blood pressure measurement, the blood pressure was significantly lower for the non-smoking period than during the smoking-period. Nighttime blood pressure did not differ significantly between the 2 points. The 24-hour heart rate was significantly lower during the non-smoking period. The heart rate was significantly lower both during day and at night during the smokers-period, compared to the non-smokers smokers period (Minami et al. 1999).

14.6.12 Impact of smoking on the electrodermal activity Smoking increases the arousal in electro dermal activity (Morris and Gale, 1993; Epstein et al. 1990).

14.6.13 Influence of oral contraception on HRV, BD, RSA Rabeleo et al. examined 150 women (age 24 Âą 2) and divided them into 4 groups, in accordance with their physical activity and the use of oral conception. For shannon entropy and conditional entropy no significant difference was found between the groups (Rebelo et al. 2011). A study was conducted on 17 healthy women (19-31 years) who, for the last 6 months have consistently taken oral birth control. This study showed no significant differences between the low hormonal levels while not taking oral contraception and high hormonal levels during oral contraception. For the HRV parameters, systolic and diastolic blood pressure no significance was found(Teixeira et al. 2015). Another study showed no significant differences between RSA and the use of oral contraception, relative to the inactive pill phase (Wisslead et al. 2012).

14.6.14 Length, BMI/weight in relationship with HRV A negative correlation was found between: length and lower heart rates and between BMI and rMSSD. An increase in weight and BMI was associated with increased sympathetic activity (LF norm, LF/HF) and low parasympathetic activity (HF n. U.) (Lutfi and Sukkar, 2011). Some authors have found no connection between BMI and HRV (Farah et al. 2013; Windham et al. 2012).

14.6.15 Relationship BMI with blood pressure and heart rate (BD) Increase of BMI is related to an increase of the heart rate, systolic and diastolic blood pressure (Martins et al. 2003).

14.6.16 Correlation between BMI, BD, HRV Shekharappa et al. (2011) compared 117 healthy (obese) men (BMI of 30 kg/m2) with 110 healthy men (BMI between 18.50 â&#x20AC;&#x201C; 24.99 kg/m2). A significant increase in heart rate, systolic and diastolic blood pressure was found with obese men. A positive correlation was found between BMI and heart rate, systolic and diastolic BD, average BD and heart rate.

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14.6.17 Relation BMI and EDA BMI lowers the number of non-specific fluctuations (NSFs) but there was no significant correlation found with SCL, SCR (Kronholm et al. 1993; Doberenz et al. 2011).

14.6.18 Relationship and BMI and body fat% HR Peterson et al. reported a decline in various non-electro dermal indicators in the activity of the ANS with an increase in body fat percentage and a higher BMI (Doberenz et al. 2011).

After taking a beta blocker, significant correlations were found between: body fat percentage and variations R-R intervals (r= -03., p < 0.03), heart rate (r= 0.30, p < 0.03), plasma concentrations of norepinephrine (r= -.30, p < 0.05) and epinephrine (r= -.49, p < 0.001) and the latency period (r= 0.39, p < 0.01) (Peterson et al. 1988).

14.6.19 Ambient temperature and SCL Ambient temperature has no correlation with SCL (Turpin et al. 1983). Doberenz et al. (2011) showed that there is a significant relationship between ambient temperature and an increase in the number of non-specific fluctuations. Ambient temperature has no effect on the other electro dermal screening parameters.

14.6.20 Relationship time and EDA Electrodermal activity is the lowest in the morning (Doberenz et al. 2011; Mirรณ et al., 2002).

14.6.21 Relationship HRV day and night When looking at the HRV indices on the basis of spectral and time-domain methods, a significant difference is noticed between day and night on the HRV indices (Raemakers et al. 1998, Yamaski et al., 1996).

Nota: The following figures shows an example of possible interrelationships that can be examined.

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Age

HR

Gender

Status

BD

HRV

BMI

SCL

Each variable can be connected to another variable

Figure 13: Relationship between HRV with age and gender

RSA

HR

PEP

HRV

SCL

BD

Respiration Rate Figure 14: Possible interrelationships between the different variables

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14.7 Addendum 7: Hardware VU-AMS 5fs and software VU-AMS, VUDAMS, Kubios.

1. Hardware VU-AMS 5fs The VU-AMS 5fs (size 32 mm x H x L126 B69; weight with battery and leads 260 g) uses in total 7 Kendall ARBO H98SG electrodes for recording the ECG (3 lead electrodes) and the ICG (4 lead electrodes). The recording of SCL is done by 2 lead electrodes. In the device, an on-board compact flash card is active that can store up to 4 GB of data. The storage capacity allows a continuous ECG recording [sampled at 1000 Hz with 16 bit resolution] up to 72 hours.

2. Software VU-AMS 5fs For installing and updating the specialized software (Table 14) for the VU-AMS device one can use the free downloads through the website of the VU-AMS (At: http://www. Vu-ams.

Nl/support/previous-ams-versions/download software). The software was tested on the operating system Windows XP (compatible with Windows 9 x/NT/2000/Vista/7). An overview of the channels of the VU-AMS 5fs can be found in Table 15.

Table 14: Software VU-AMS 5fs.

Software AmsConfigure AmsMerge AmsPreProcessor

Inhoud Communication program for the configuration of the VU-AMS 5fs device.

Program for the merge of several 5fs files Program for the preparation of 5fs files to an AMS format, export of ASCII, EDF or Biopac

AmsQRS

Program for the automatic score of R-peaks in the rough ECGsignal and visual inspection of the final IBI time series. AMSRES

AmsGraph AMSGRA

Program for the visual inspection of rough signals.

Program for the graphic display of the VU-AMS registration.

AMSASC

Program for converting binary VU-AMS files to an ASCII-format

AMSRES

Program for viewing the respiration score (dZ) data.

AMSIMP

Program for viewing the ICG data.

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Table 15: Channels VU-AMS 5fs Channels

Sampling interval

Min. physical value

Max. physical value

ECG 1 [msec] (1000 [Hz]) -20.0 [mV] 20.0 [mV] dZ 1[msec] (1000 [Hz]) -1.0 [Ohm] 1.0 [Ohm] MZR 1 [msec] (1000 [Hz]) -5.0 [g] 10.0 [g] MXR 1 [msec] (1000 [Hz] 1 [msec] (1000 [Hz] 10.0 [g] SCL 100 [msec] (10 [Hz] 0.0 [uS] 95.0 [uS] BAT 100 [msec] (1 [Hz] 0.0 [V] 5.0 [V] ZO 4 [msec] (250 [Hz] 0.0 [Ohm] 37.0 [Ohm] MYA 1000 [msec] (1 [Hz] 0.0 [g] 15.0 [g] MYR 1[msec] (1000 [Hz] -5.0 [g] 10.0 [g] dZ/dt 1[msec] (1000 [Hz] -500.0 [Ohm/s] 500.0 [Ohms/s] ECG: electrocardiogram; dZ: delta Z; Z0 is the thoracic impedance, dZ is the change; MZR: movement in Z direction; MXR: movement in X direction; SCL: skin conductance level BAT: battery; MYA: movement in Z direction (AC component); dZ/dt (1ste derivative of Z0), Min: minimum, Max: maximum.

3. Software program VU-DAMS (v3.9) VU-DAMS* is a data analyzer and management software program developed by the Free University of Amsterdam for analyzing: heart rate variability (Time domain and Frequency domain); respiration score; impedance cardiography and electrodermal activity. Both the software and the manual of the VU-DAMS v3.9 can be downloaded for free over the internet through the website of the VU-AMS (At: http://www. Vu-ams. Nl/ fileadmin/user_ upload/manuals/VU-DAMS_manual_v1.2. Pdf.).

In the VU-DAMS program there are 8 different tabs available for processing the VU-AMS data. * for the synopsis software VU-DAMS (v3.9) the document 'Data analysis and management software (DAMS) for the free University Ambulatory Monitoring System (VU-AMS) was used.

Manual version 1.2 dd31/07/2015 (written for VU-DAMS v3.9)'.

3.1 Inspection of the raw data In inspect data all data (MYA, MZR, MYR, MXR, Z0, dZ, SCL, ZDT, ECG) that have been recorded in the VU-AMS device are listed. All these data are shown in time series. The bottom bar in this tab represents the entire ECG recording time.

3.2 R-Peak detective and correction In this tab, manual adjustments and corrections of the R-tops in the ECG can be made. R-R intervals are automatically checked by a beat detector and colored lines are subsequently plotted on top of the raw ECG signal (R-R tops). By using yellow (possible suspect IBI's) or red (highly suspect IBI's) color codes, the need for correction is shown. The blue color code represents the R-tops which, according to the automatic beat detector, have been approved.

The QRS detector software automatically performs 'detect R-peaks', three separate analyses on the ECG signal namely: an artefact detector, R-peak detector, suspicious beats (suspect beats) detector.

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3.3 Labeling of the data Through this tab the data collected by the VU-AMS device can be divided into time periods for further analysis. This division is called 'labels'. The purpose of the tags is to show an average value for each available parameter (HR, PEP, RSA, HRV, SCL) and this for each experimental condition.

3.4 frequency domain analysis For the whole of the ECG recording in this tab the IBI, MYA(g), LF and HF power (ms2), and the heart rate (sl/min) are shown. For the HRV spectral analysis, the IBI time series for each label are interpolated using a cubic spline, with a resulting pixel dimension of 4 Hz. In a cubic spline interpolation various observations are being arranged according to the size of an independent variable and connected to a piece of a cubic (= graph of a 3rd degree function) (Van de Woestyne, 2015). The signal is divided into overlapping periods of 256 seconds with each 1024 data points. Between two consecutive periods there is an overlap of 128 seconds.

Periods that contain intervals which take longer than 5 seconds without IBI are being removed. Data which are missing from the last period are filled with zero's. Each period of 1024 data points is being processed through a ‘smoothness prior matrix (λ 500)' where a 'Fast Fourier Transform analysis' in a quadratic window is applied. The ‘Welch's periodogram method' is applied to determine the power value (displayed in ms2) of each 1024 data points.

Both for the low frequency bands (0.04-.15 Hz) and the high frequency bands (0.15-.40 Hz).

To determine the low frequency power, an ECG recording time of at least 4 minutes is required.

3.5 Impedance score This tab shows the impedance cardiogram (ICG) per label and is divided into four windows.

The left window is subdivided (ICH complex, marker positions, calculated parameters) and shows different values of the ICG. The right window shows the graph of the ECG and dZ/dt.

At the bottom, the IBI of the whole ECG recording are visible. At the top of the right window (dZ/dt), the B-point, C-point and the X-point are being determined. At the bottom of the right window the Q-wave onset is shown.

B-point: this point lies just before the beginning of the slope (deflection) of the incisura.

Normally ithe B-point of the dZ/dt golf is found under the 0 baseline, sometimes is also above the 0 baseline. B-point represents the onset of the left ventricular ejection.

C-point: this is the highest point in the dZ/dt golf. At this point, the speed of the ejection is on its maximum and the impedance at its minimum. Because the graph of the ICG is displayed in a reverse polarity, the highest peak in the dZ/dt golf represents the lowest impedance. The reason of the reverse polarity is related to the readability and interpretation of the chart, similar to reading and interpreting an ECG. X-point: this is the lowest point in the dZ/dt golf. The X-point represents the conclusion of the aortic valve at the end of the left ventricular ejection. If there are 2 equal points present, the 2nd point is taken for the indication of the X-point.

Q-wave onset: this point lies on the transition from the beginning of the fall of the Q wave. It represents the start of the ventricular depolarization of the interventricular septum.

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3.6 Respiratory sinus arrhythmia (RSA) This tab gives an overview of dZ, heart rate, RSA and the respiration rate. The VU-DAMS has three detection systems to detect artifacts (clipping dZ, irregular respiration, irregular IBI). If necessary one can manually mark additionally artifacts here. At the top of the window dZ, the beginning of the expiration (blue triangle) and beginning of the inspiration (red triangle) are shown. The purple bar reflects the overlap of the inspiration phase with the expiration phase. In the cardio tachogram the shortest IBI (red) during the inspiration and the longest IBI (blue) during the expiration are determined. The RSA window gives an overview of the time series of the RSA values over the successive breathing, expressed in milliseconds. In the window of the respiratory frequency, the successive breaths per minute are shown.

3.7 Skin conduction Per label in this tab the label information and SCL is given at the top of the label. There are two charts shown (per label), on top the FILT-SCL and at the bottom of the heart rate.

Artifacts can be indicated manually in the bar below the window of the heart rate chart.

3.8 Label information This is the last tab in the software program VU-DAMS. Here one can find a detailed overview of all parameters (horizontal) with corresponding values (vertical). The data can be exported to other software programs (e. G. Excel, Kubios).

4 Software program Kubios (v2.2) Kubios* is a software program developed by the University of Kuopio East Finland for analyzing heart rate variability in the time domain, frequency domain and not linear analysis.

Both the software program Kubios version 2.2 and the manual Kubios HRV (version 2.2) user's Guide, June 5, 2014 can be downloaded for free via the internet (At: http://kubios-hrv.

Software. Informer. Com/2.2/; http://kubios. uef. fi). The current version 2.2 is largely similar to version 2.1, with some improved adjustments. The input data support was extended (Garmin FIT les, EDF+ , update Biopac), and multiscale entropy was additionally included in the nonlinear analyses. The software program is supported by Windows, Linux, Mac Os (new for version 2.2). In the data overview of Kubios v 2.2, the window can be divided in 4 compartments, as there are: RR series options; data browser; options for analysis; results overview. * For the synopsis of software Kubios was ' user's Guide HRV version 2.2, June 5, 2014' used.

4.5 RR series options Here one can execute an automatic artefact correction of the R-R interval series depending on the desired level (none, very low, low, medium, strong, very strong, custom). Furthermore, at ' samples for analysis ' the start time and the duration of one or more labels can be selected for HRV analysis per label. The option 'remove trend components' can be used for removing troubling low frequency components from the RR interval series. In this option one can choose for 'detrending options' to remove the 1st to the 3rd order of the linear trend or ‘smoothness priors'. At the last option the value of Lambda (λ) (fc = 0.035 Hz) can be set manually. By default, Kubios set the value at λ 500.

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

The RILHOU-RCT applied standard at the automated artefact correction the level Low.



The start and stop time of the obtained data through the VU-AMS of label 2, 4 and 5 in the software program VU-DAMS are recalculated via Excel to be able to enter in Kubios v2.2. This is necessary because Kubios v2.2 standard starts with a starting hour 00:00:00 which is not in accordance with the start hour of the data obtained through the VU-AMS. The start hour is introduced in 'per label samples for analysis', each with a time length of 10 minutes.



By ‘Remove trend components' 'smoothness priors' is indicated and the default setting of the Λ value 500 retained.

4.6 Analysis options By default, Kubios has determined the frequency bands (VLF = 0-.04 Hz; LF = 0.04-.15 Hz; HF = 0.15-04 Hz). A cubic spline interpolation is being used where the sampling rate is taken on 4 Hz. For the HRV spectrum analysis 2 methods are used: the Welch's periodogram method (FFT spectrum) and an auto regression method (AR spectrum). In the FFT (Fast Fourier Transform) spectrum the default values are set on 256 seconds and respectively 50% For the auto regression spectrum, the default value, for the order, is set on 16 and one can make use of a ‘spectral factorization' option. All of these default values can be adjusted manually.

4.7 Data browser This window displays both the ECG (mV) as the RR (s) intervals graphically. In the ECG, each R-peak is marked with a red +. This marker can be moved, deleted, or added manually 'if necessary', If one wishes an ECG print (pdf) can be downloaded in the data browser.

5. Results overview HRV analysis is divided into time-domain, frequency domain and non-linear analysis . 

Time-domain: In this analysis, the following parameters are displayed: mean RR, SDNN, mean HR rMSSD, pNN50, SD, HR, NN50 Also geometric analysis, RR triangular index and TINN, are displayed..







Frequency domain: In this domain a FFT and AR spectrum analysis is used for following parameters: VLF, LF, HF. For these 3 bands, both the peak and power (ms2, %, n. U.) are displayed. The total power (ms2) and LF/HF power (ms2). In the FFT analysis VLF power is not expressed in normal units (n. U.). In the AR spectrum analysis there is no VLF peak (Hz).

In the ECG, the respiration frequency is derived from the amplitude of the R-wave.

The ECG derived respiration (EDR) is displayed under spectrum Y-limits, and expressed in Hertz (Hz).

Non-linear analysis: this analysis can be divided into three groups: poincare plot (SD1, SD2), recurrence plot (Lmean, Lmax, REC, DET, ShanEn), other (ApEn, SampEn, DFA α1 and α2, D2).

211

14.8 Addendum 8: Socio-demographic data description 14.8.1 Data collection experiment/Sham en follow-up The data collection for the experimental group (N = 20) and Sham group (N = 20) took place at FICO between the period of 13/06/2016 - 8/06/2015. The data collection for the experimental- and Sham follow-up took place between 20/06/2015 - 4/07/2015. In the experimental group 8 sessions took place in the morning, 9 sessions in the afternoon and 3 sessions in the evening. For the Sham group it concerned 4 sessions in the morning, 12 sessions in the afternoon and 4 sessions in the evening. The follow-up of the experimental group took place 6 to 9 days after the experiment (Mean: 7.7; Mode: 7; Median: 7; S: 0.54).

The Sham follow-up on average took place 6 to 8 days (Mean: 7.15; Mode: 7; Median: 7; S: 0.48).

14.8.2 Nationality Both groups existed of 19 subjects with Belgian nationality and 1 subject with Dutch nationality.

14.8.3 Gender The experimental group consisted of 9 men and 11 women. The Sham group consisted of 7 men and 13 women.

14.8.4 Age The average age in the experimental group was 30.9 (Mode: 28.11; Median: 30.08; S: 6.60; Range: 20-48). The average age for the men was 33.06 (Median: 31.11; S: 7.78), while for the women it was 29.07 (Median: 30.06; SD 3,23) was. The average age in the Sham group was slightly higher (Mean: 31, 9; Mode: 28.06; Median: 32.09; S: 6.37; Range: 23-44), compared with the experimental group. In the Sham group the average age for men was similar to the one of the experimental group (Mean: 33.06; Median: 33.10; S: 6.67). The average age of the women was slightly higher in the Sham group, compared to the experimental group (Mean: 31.10; Median: 30.04; S: 6.01).

14.8.5 BMI In the experimental group the average BMI was lower (Mean: 23.02; Mode: 24.10; Median: 22.45; S; 2.77; Range: 12-29.1), compared to the Sham group (Mean: 23.09; Mode: 24.10; Median: 23.15; S: 2.79; Range: 19.4-27.7). The average BMI was slightly lower for the men in the experimental group (Mean: 23.48; Mode: 24.10; Median: 24.10; S: 1.98; Range: 21.725), compared with the men in the Sham group (Mean: 23.49; Mode: 27; Median: 23.50; S: 3.13; Range: 19.4-27). The average BMI was lower for the women in the experimental group (Mean: 22.64; Median: 21.60; S: 3.44; Range: 19.3-29.1), compared with the women in the Sham group (Mean: 22.87; Median: 22.30; S: 2.81; Range: 19.6-27.7). The average BMI for the men was higher in both groups, compared with the women.

212

14.8.6 Civil status In the experimental group there were 8 cohabiting (1 legally cohabiting), 6 singles, 5 married and 1 divorced subject. Compared with the experimental group there were in the Sham group more cohabiting subjects (12), less married couples (4), less people who live alone (3) and an ex aequo for divorced subjects (1).

14.8.7 Number of hours of sports The total number of hours of sport in the experimental group amounted 53u30 (Mean: 2.67; Median: 2; S: 2.67). In the Sham group the total number of hours of sports was higher (58u50), compared to the experimental group (Mean: 2.93; Median: 3; S: 1.66).

14.8.8 Shift patterns In the experimental group, there were 5 subjects who worked in 24 hours shift pattern and 2 subjects who either worked with an early or late shift pattern. In the Sham group, the number of subjects that worked in a shift pattern was lower (total 5 = 4 subjects with 24 hours shift; 1 subject with either early or late shift), compared with the experimental group.

14.8.9 Smoking In the experimental group more smokers were present (total = 6, 3 men and 3 women), compared to the Sham group (1 male smoker). The average number of cigarettes per day was higher in the experimental group (Mean: 6.67; S: 2.81), compared to the Sham group (Mean: 3).

14.8.10 Number of glasses of alcohol a week In the experimental group there were 17 subjects (8 men; 9 women) who consumed alcohol on a weekly basis. The average number of glasses of alcohol in this group was higher with men (Mean: 7.56; Median: 6; S: 4), compared to women (Mean: 3.55; Median: 2; S: 3.60). In the Sham group there were less subjects who consumed alcohol on a weekly basis (5: men, 9: women), compared to the experimental group. Compared to the experimental group, the average number of glasses of alcohol per week was lower in the Sham group with men (Mean: 2.8; Median: 2; S: 1.17) and higher with women (Mean: 3.89; Median: 3; S: 2.28).

Compared to men, an average in the Sham group of women consumed a larger number of glasses of alcohol a week.

14.8.11 Allergy In the experimental group there were fewer reports of allergy (total= 7; 5 women; 2 men), compared with the Sham group (total= 8; 7 women, 1 man). In both groups women showed more allergies, compared to men. In the experimental group following allergies were registered: hay fever (2 pp*); grass pollen (1 pp, man); pollen (1 pp, man); nickel (1 pp); dust mites (1 pp); cow's milk (1 pp); goat's milk (1 pp); casein (1 pp); cats (1 pp); maize (1pp), penicillin (1 pp); Cortisone (1 pp).

213

In the experimental group there were fewer reports of allergy (total = 7; 5 women; 2 men), compared to the Sham group (total = 8; 7 women, 1 man). In both groups women showed more allergies, compared to men. For the Sham group following allergies were registered: dust mites (3 pp, 1 man); pollen (3 pp, 1 man), cats (2pp, of which 1 man); nickel (2 pp); Pineapple (1 pp); gluten (1pp); lactose (1 pp); dogs (1 pp, man); horses (1 pp, man); iodine (1 pp); dexmethasone (1 pp); penicillin (1 pp); diphenhydraminehydrochl (1 pp).

*pp= subject

14.8.12 Categorical scale related to the experience of pain In the experimental group there were in total 6 subjects (5 females, 1 male) who scored more than 0 (no pain) on the perception of pain on the categorical scale between 0-10. 3 subjects assessed their pain between the 3-4. Two subjects assessed the pain experience on 3, one test subject (man) assessed his pain on 4. In the Sham group, there were a total of 8 subjects (7 women, 1 man) who assessed more than 0 on the perception of pain on the categorical scale. 4 subjects assessed the pain experience between 1-6. Four subjects assessed their pain experience as follows: 1, 3, 4 and 6 (male).

14.8.13 Use of non-conventional medicine and/or alternative form In both groups only one women consulted so-called non-conventional medicine or an alternative form (experimental group 2 pp; Sham group 6 pp). In both groups osteopathy was consulted (experimental group 1 pp; Sham group 4 pp). In the experimental group one subject used an infrared cabin (1pp). In the Sham group there were 3 subjects who practiced yoga.

14.8.14 Supplementation of Ί3 fatty acids Only in the Sham group was Ί3 fatty acids taken as supplementation by 3 women.

14.8.15 Medication Both groups used hormonal anticonception. In the experimental group there were less women taking hormonal anticonception (4 pp), compared with the Sham group (7 pp).

Inventory names medicines: Meliane appeared only once in both groups. In the experimental group Desorelle 20 (2 pp) and Gracial was used. In the Sham group Desorelle 30; Gestodelle; Nora 30; Yasminelle and a hormonal coil (2 pp) was used as hormonal anticonception.

Other medication: in the experimental group 1 person (man) was taking Rupatall (H1antihistamines) and Mometasone Sandoz nasal spray (allergy). Folic acid (vitamin B11) was taken by 1 female subject.

214

14.8.16 Ambient temperature For the experimental group the average ambient temperature was 24.17 °C (Mode: 25; Median: 24; S: 0.87), while for the Sham group the average was 24.72 °C (Mode: 25; Median: 25; S: 0.75). The average ambient temperature for the experimental follow-up was higher compared to the experiment (Mean: 25.5; Mode: 25; Median 25; S: 2.01), but was slightly lower compared to the Sham follow-up (Mean: 25.85; Mode: 25; Median: 25, S: 2,55).

14.8.17 Relative humidity percentage The average relative humidity percentage was slightly higher in the experimental group (Mean: 36.4; Mode: 34; Median: 35; S: 3,48), compared to the Sham group (Mean: 36.35; Mode: 34; Median: 35.50; S: 3.02). This was characteristic for both groups during the followup (Expi. follow-up: Mean: 41.7; Mode: 0.44; Median: 42.5; S: 3.59; Sham follow-up: Mean: 40.95; Mode: 41; Median: 41; S: 3.9). For both groups the average relative humidity rate is higher during the follow-up, compared to the experiment/Sham.

215

14.9 Addendum 9: Measure of central tendency and dispersion: HRV, SCL, RSA, respiration rate, blood pressure (Note: the meanings of the abbreviations in the tables are only once presented per page.)

14.9.1 HRV: Time domain Table 16: Measure of central tendency and dispersion for average heart rate [V] Average HR (bpm) [V]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

65,19

63,52

↓

68,63

↑

62,83

62,17

↓

63,90

↑

Median

62,20

64,52

↑

69,15

↑

64,79

62,47

↓

65,31

↑

70%

↓

65%

↑

60%

↓

60%

↓

* CVi CVg

0,18

§ & SD SEM (i) SEM (g) CI: Lower (i) CI: Uper (i) CI: Lower (g)

0,05

0,10

0,16

0,13

-3,47

7,58

-2,55 11,57

10,33

5,29 ↓

8,91

60,12

0,68 2,31 63,02 65,69 59,00

1,49 1,99 63,99 69,83 64,73

CI: Uper (g)

70,25

68,05

Skewness

0,26

-0,10

Kurtosis

-0,75

-0,94

2,59

0,17

0,02

0,05

0,15

0,16

-0,90

2,27

-1,06 ↓

10,41

9,38

1,71 ↓

10,12

58,27

0,34 2,10 61,83 63,17 58,06

0,72 2,26 61,96 64,78 59,47

72,54

67,39

66,28

68,34

-0,06

-0,19

-0,03

-0,14

0,98

-0,90

-1,03

-0,57

2,33

↓

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intraindividual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

216

Table 17: Measure of central tendency and dispersion for average IBI [V] Average IBI (ms) [V]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

953,80

976,27

↑

893,61

↓

986,34

994,56

↑

968,96

↓

Median

974,96

937,35

↓

869,48

↓

931,35

966,43

↑

924,16

↓

70%

↑

60%

↑

60%

↑

* CVi CVg

0,05

§ &

0,05

0,10

1,16

0,13

0,29

-4,30

2,36

SD

165,90

SEM (i) SEM (g)

158,89

↓ 0,18

-6,31 ↓

11,03 37,10

60%

115,97

0,02

0,05

0,16

0,17

0,14

-1,24

0,83 ↓

174,40

20,87

158,83

-1,76 ↓

5,26 39,00

↓

166,01 10,70

35,53

25,93

35,52

37,12

CI: Lower (i)

943,42

882,80

980,14

956,68

CI: Uper (i)

986,66

964,61

1000,75

998,61

CI: Lower (g)

881,10

906,64

842,78

909,90

924,95

896,20

CI: Uper (g)

1026,51

1045,91

944,43

1062,78

1064,17

1041,72

Skewness

-0,13

0,24

0,21

0,32

0,18

0,27

Kurtosis

-0,82

-1,20

0,38

-0,70

-1,05

-0,40

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intraindividual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

Table 18: Measure of central tendency and dispersion for rMSSD [V] rMSSD (ms) [V]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

66,93

67,54

↑

43,44

↓

56,45

64,12

↑

63,16

↑

Median

51,07

55,89

↑

38,75

↓

43,77

45,64

↑

49,98

↑

60%

↑

65%

↓

75%

↑

70%

↑

* CVi

0,24

0,41

0,60

0,64

§

26,03

&

0,90

CVg

SD

0,99

65,93

SEM (i) SEM (g)

14,74

CI: Lower (i) CI: Uper (i)

40,69

↓

0,12

0,16

0,76

0,71

-15,29

20,15

12,74

-35,09

13,59

11,89

27,97

3,64

5,01

9,10

6,25

60,11

45,38

0,66

↓

37,53 8,39

48,64

↑

44,92

1,56

2,19

10,88

10,04

57,22

55,52

74,37

65,00

63,35

64,09

CI: Lower (g)

38,04

49,70

31,18

40,00

42,80

43,47

CI: Uper (g)

95,83

85,37

55,70

72,90

85,44

82,84

Skewness

0,24

0,29

0,17

0,34

0,38

0,29

Kurtosis

11,11

0,54

0,24

1,71

4,07

0,42

↑

217

Table 19: Measure of central tendency and dispersion for pNN50 [K] pNN50 (%) [K]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

29,62

33,03

↑

19,23

↓

26,72

27,57

↑

33,24

↑

Median

25,90

31,94

↑

18,01

↓

22,04

19,02

↓

28,24

↑

60%

↑

75%

↓

55%

↑

75%

↑

* CVi

0,20

0,42

0,58

0,99

§

-411,58

&

11,51

CVg

SD

0,76

22,41

SEM (i)

19,30

↓

0,12

0,21

0,78

0,78

-24,73

47,95

36,16

-35,07

3,16

24,40

19,03

1,41

2,30

4,31

4,26

CI: Lower (i)

28,56

CI: Uper (i)

34,08

SEM (g)

5,01

0,85

↓

22,65

21,49

↓

25,79

0,71

1,43

4,81

5,77

19,92

25,75

27,17

28,93

28,54

32,79

5,06

CI: Lower (g)

19,80

24,57

10,89

16,80

18,15

21,94

CI: Uper (g)

39,80

41,48

27,57

36,65

36,99

44,55

Skewness

0,17

0,06

0,06

0,21

0,40

Kurtosis

-0,82

-0,78

-0,58

-0,89

-0,95

↑

0,19 -1,52

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intraindividual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

Table 20: Measure of central tendency and dispersion for SDNN [V] SDNN (ms) [V]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

66,78

80,77

↑

64,19

↓

63,49

78,09

↑

71,13

↑

Median

58,35

81,00

↑

59,21

↑

55,33

59,73

↑

68,92

↑

75%

↑

60%

↑

85%

↑

65%

↑

* CVi CVg

0,51

0,22

0,25

0,38

0,52

0,43

0,17

0,16

0,55

0,46

§

33,87

5,43

25,68

15,48

&

20,95

-3,88

23,00

12,04

SD

34,39

SEM (i) SEM (g)

31,00 3,70

7,69

↓

33,15

↓

27,20

3,70

43,03 2,64

6,08

↑

32,68

↑

2,48

6,93

7,41

9,62

7,31

CI: Lower (i)

66,51

58,22

65,61

62,46

CI: Uper (i)

81,03

72,74

75,97

72,16

CI: Lower (g)

51,70

67,18

49,66

51,56

59,23

56,81

CI: Uper (g)

81,85

94,35

78,71

75,41

96,94

85,45

Skewness

0,25

-0,01

0,15

0,30

0,43

0,07

Kurtosis

3,29

-0,27

0,32

1,94

5,07

1,58

218

14.9.2 HRV: Frequence domain Table 21: Measure of central tendency and dispersion for LF ms2 FFT [V] Experimental group

2

LF ms FFT [V]

t0

t1

Sham group t2

t0

t1

t2

Mean

1155,84

1646,34

↑

1078,80

↓

1464,06

1954,58

↑

1381,91

↓

Median

694,88

1597,71

↑

967,94

↑

938,12

1200,25

↑

1023,69

↑

65%

↑

50%

↑

75%

↑

65%

↑

* CVi CVg

1,06

§ &

0,48

0,44

0,74

0,84

4,12

29,41

42,44

SD

1225,39

SEM (i) SEM (g) CI: Lower (i) CI: Uper (i) CI: Lower (g)

1224,83

1,43

-6,67 ↓

910,32

0,31

1,25

1,02

3,57

40,94

33,50 ↓

2093,01

-5,61

2449,05

↑

1402,97

618,79

149,26 273,88 1108,55 1693,63 1109,53

CI: Uper (g)

1692,89

2183,14

1477,76

Skewness

0,38

0,04

0,12

0,25

0,31

0,26

Kurtosis

4,30

0,35

1,10

13,73

5,50

10,02

274,00

109,97 203,55 901,77 1332,87 679,83

0,24

546,76

91,07 547,63 1530,83 1887,23 881,23

98,21 313,71 1230,50 1230,50 767,04

2381,36

3027,92

1996,79

468,01

↓

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

Table 22: Measure of central tendency and dispersion for LF ms2 AR [K] Experimental group

2

LF ms FFT [V]

t0

t1

Sham group t2

t0

t1

t2

Mean

1322,77

1691,75

↑

1129,57

↓

1363,17

1945,35

↑

1437,11

↑

Median

697,67

1542,73

↑

998,97

↑

902,12

1059,00

↑

1037,70

↑

60%

↑

55%

↓

75%

↑

70%

↑

* CVi CVg

1,11

0,49

0,48

0,73

0,85

1,30

0,29

0,28

1,22

0,99

§

2,62

21,00

3,07

39,34

&

27,89

-14,61

42,71

5,42

SD SEM (i) SEM (g) CI: Lower (i) CI: Uper (i) CI: Lower (g)

1464,94

1236,26

↓

963,43

1766,14

2369,85

↑

1416,90

680,73

164,25 276,44 1185,32 1829,20 1149,94

CI: Uper (g)

1964,81

2233,57

1551,81

Skewness

2,13

0,73

1,13

3,50

2,29

2,83

Kurtosis

4,20

0,17

1,31

13,66

4,74

8,98

327,57

130,50 215,43 970,40 1481,94 707,32

↓

589,12

107,88 529,91 1442,81 1865,71 906,72

88,30 316,83 1227,07 1573,20 816,12

2137,21

2983,99

2058,09

394,92

↓

219

Table 23: Measure of central tendency and dispersion for LF n.u.FFT [K] LF n.u.FFT [K] Mean Median

Experimental group t0 50,06 46,55

* CVi

t1

Sham group t2

52,81

↑

60,28

↑

53,16

↑

62,92

↑

65%

↑

70%

↑

0,17

0,23

0,28

0,29

§

9,69

&

5,50

CVg

SD

0,30

15,02

SEM (i) SEM (g)

3,36

CI: Lower (i) CI: Uper (i)

t0

14,62

↓

53,91 53,03

t1

t2

57,50

↑

52,42

↓

56,72

↑

53,65

↑

65%

↑

50%

↓

0,14

0,14

0,25

0,34

28,00

18,32

-0,24

20,42

6,67

-2,75

17,45

1,90

2,78

3,27

3,90

47,71

49,72

0,29

↑

15,84 3,54

14,27

↓

17,73

1,71

1,65

3,19

3,96

52,34

49,93

55,16

60,62

59,07

56,40

CI: Lower (g)

43,47

46,40

52,63

46,96

51,25

44,65

CI: Uper (g)

56,64

59,22

67,93

60,85

63,76

60,19

Skewness

0,23

-0,02

-0,15

0,06

0,05

-0,07

Kurtosis

-0,57

-0,15

-1,41

-0,45

-0,18

-0,62

↑

Table 24: Measure of central tendency and dispersion for LF n.u. AR [K] LF n.u. AR [K]

Experimental group t0

t1

Sham group t2

t0

Mean

50,17

53,84

↑

Median

49,95

53,48

↑

64,63

↑

60%

↑

75%

↑

* CVi

60,10

↑

52,01 53,84

0,14

0,21

0,24

0,27

§

27,44

&

7,32

CVg

SD

0,35

17,62

SEM (i)

13,15

↓

58,54

52,40

↑

55,88

↑

52,56

↓

60%

↑

55%

↑

0,15

0,21

0,32

30,74

20,06

-0,81

19,80

12,55

0,75

15,94 2,63

2,94

3,56

CI: Lower (i)

48,84

CI: Uper (i)

55,16

3,94

t2 ↑

0,13

1,61

SEM (g)

t1

0,36

↓

18,84

12,06

↓

16,52

1,57

1,75

2,70

3,69

49,99

52,19

48,78

60,28

58,35

55,63

4,21

CI: Lower (g)

42,45

48,07

53,12

43,75

53,25

45,16

CI: Uper (g)

57,89

59,61

67,09

60,27

63,82

59,64

Skewness

0,01

0,03

-0,28

0,04

0,22

-0,01

Kurtosis

-1,12

0,13

-1,39

1,87

-0,50

-0,60

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

220

Table 25: Measure of central tendency and dispersion for HF ms2 FFT [V] 2

HF ms FFT [V]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

1296,09

1438,54

↑

725,51

↓

1199,30

1414,92

↑

1709,76

↑

Median

802,72

1138,47

↑

460,39

↓

649,38

574,07

↓

845,83

↑

65%

↑

65%

↓

60%

↓

70%

↑

* CVi

0,36

0,54

0,82

1,04

§

5,89

&

10,99

CVg

1,04

SD

1343,30

SEM (i) SEM (g)

300,37

1180,65

↓

0,19

0,36

1,42

1,42

-16,56

0,76

39,44

-44,02

17,98

42,56

755,97

109,46

121,32

264,00

169,04

1,25

↓

1493,42 333,94

2005,29

↑

2421,97

56,64

118,07

448,40

541,57

CI: Lower (i)

1152,78

773,02

1196,09

1223,11

CI: Uper (i)

1581,85

1248,59

1418,12

1685,95

CI: Lower (g)

707,37

921,09

394,19

544,78

536,06

648,28

CI: Uper (g)

1884,82

1955,98

1056,82

1853,82

2293,78

2771,24

Skewness

0,37

0,25

0,35

0,37

0,42

0,36

Kurtosis

1,89

-0,75

0,84

6,35

5,67

4,02

↑

Table 26: Measure of central tendency and dispersion for HF ms2 AR [K] 2

HF ms AR [K]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

1712,97

1625,89

↓

811,74

↓

1219,70

1483,04

↑

1846,35

↑

Median

884,94

1284,76

↑

546,55

↓

740,95

657,52

↓

1066,20

↑

65%

↑

65%

↓

65%

↑

85%

↑

* CVi CVg

1,54

§ & SD

0,64

0,82

1,04

5,14

-17,39

-5,08 2645,50

SEM (i) SEM (g)

0,45

591,55

1327,26

1,17

-52,61 ↓

843,82

169,22

179,99

0,18

0,39

1,34

1,21

1,57

55,95

21,59 ↓

1423,10

1986,28 54,11

318,22

51,38 ↑

2226,77

↑

133,34

296,79

188,68

444,14

497,92

CI: Lower (i)

1337,75

909,57

1245,32

1271,68

CI: Uper (i)

2001,11

1615,14

1457,42

1794,37

CI: Lower (g)

553,53

1044,19

441,92

596,00

612,52

870,43

CI: Uper (g)

2872,41

2207,59

1181,56

1843,40

2353,57

2822,28

Skewness

0,31

0,26

0,31

0,34

0,42

0,35

Kurtosis

12,91

-1,05

0,98

3,20

5,47

3,21

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

221

Table 27: Measure of central tendency and dispersion for HF n.u.FFT [K] HF n.u.FFT [K]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

49,86

47,12

↓

39,66

↓

45,98

42,44

↓

47,50

↑

Median

53,39

46,83

↓

37,03

↓

46,32

43,27

↓

46,18

↓

65%

↓

70%

↓

65%

↓

50%

↑

* CVi CVg

0,30

§ & SD

0,28

0,31

0,44

-9,36

-10,31

-5,50 14,98

SEM (i) SEM (g)

0,17

14,56

-20,46 ↓

1,89 3,35

0,34

17,44

0,17

0,16

0,34

0,37

-21,67

5,35

-7,72 ↑

15,81

2,78

14,28

3,29 ↓

1,71 3,54

17,73

↑

1,64

3,26

3,90

3,19

3,96

CI: Lower (i)

44,78

39,31

40,87

43,52

CI: Uper (i)

52,20

50,20

47,56

49,96

CI: Lower (g)

43,29

40,73

32,01

39,06

36,18

39,73

CI: Uper (g)

56,42

53,50

47,30

52,91

48,70

55,27

Skewness

-0,24

0,02

0,15

-0,02

-0,06

0,07

Kurtosis

-0,57

-0,13

-1,40

-0,45

-0,20

-0,62

Table 28: Measure of central tendency and dispersion for HF n.u. AR [K] HF n.u. AR [K]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

49,75

46,09

↓

39,83

↓

45,50

41,41

↓

47,53

↑

Median

50,01

46,50

↓

35,34

↓

46,11

44,04

↓

47,29

↑

60%

↓

75%

↓

60%

↓

50%

↑

* CVi CVg

0,35

§ & SD

0,26

0,28

0,40

-1,70

-11,75

-7,36 17,58

SEM (i) SEM (g)

0,15

13,10 1,61

3,93

0,32

-19,94 ↓

15,91

0,12

0,14

0,29

0,35

-22,98

5,34

-8,99 ↓

14,38

2,62

12,06 1,19

3,22

4,47 ↓

16,51

↑

1,46

2,93

3,56

2,70

3,69

CI: Lower (i)

44,77

39,65

41,12

43,65

CI: Uper (i)

51,07

49,94

45,78

49,38

CI: Lower (g)

42,05

40,35

32,86

39,19

36,12

40,29

CI: Uper (g)

57,46

51,83

46,80

51,80

46,69

54,76

Skewness

-0,01

-0,03

0,28

-0,04

-0,22

0,01

Kurtosis

-1,12

0,10

-1,39

-0,25

-0,52

-0,61

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

222

Table 29: Measure of central tendency and dispersion for LF/HF ratio ms2 FFT [K] LF/HF ratio 2 ms FFT [K]

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

1,26

1,35

↑

2,07

↑

1,53

1,75

↑

1,52

↑

Median

0,87

1,14

↑

1,73

↑

1,15

1,32

↑

1,16

↑

65%

↑

70%

↑

65%

↑

50%

↓

* CVi CVg

0,76

§ & SD

0,57

0,63

0,67

966,34

113,96

7,46 0,96

SEM (i) SEM (g)

0,35

0,21

0,85

0,80

64,73 ↓

1,39

0,10

0,21

0,28

0,25

0,81

0,90

1819,31

7,61

14,18 ↑

1,23 0,28

1,42

-0,79 ↑

1,36

0,10

0,08

0,19

0,31

0,32

0,30

CI: Lower (i)

1,10

1,25

1,44

1,36

CI: Uper (i)

1,51

2,08

1,85

1,69

CI: Lower (g)

0,84

0,98

1,46

0,99

1,13

0,92

CI: Uper (g)

1,68

1,73

2,68

2,07

2,37

2,12

Skewness

0,41

0,25

0,24

0,31

0,30

0,26

Kurtosis

3,36

1,88

-0,72

3,86

7,04

5,86

↑

Table 30: Measure of central tendency and dispersion for LF/HF ratio ms2 AR [K] LF/HF ratio 2 ms AR [K]

Experimental group t0

Mean

1,36

Median

1,00

* CVi

t1

Sham group t2

1,37

↑

1,15 60%

1,97

↑

1,49

↑

1,84

↑

1,17

↑

75%

↑

0,28

0,50

0,59

0,67

§

2741,82

&

1,27

CVg

SD

0,82

1,12

SEM (i)

0,81

↓

t2

1,71

↑

1,27

↑

1,11

= ↓

60%

↑

50%

↓

1,49

0,23

0,67

0,91

103,93

2067,72

4,22

45,26

15,47

0,36

1,31 0,18

0,18

0,29

CI: Lower (i)

1,20

CI: Uper (i)

1,53

0,25

t1

0,21

0,08

SEM (g)

t0

0,70

↑

1,05

1,15

↑

1,35

0,07

0,07

0,26

0,30

1,30

1,45

1,34

2,02

1,75

1,63

0,23

CI: Lower (g)

0,87

1,02

1,39

1,03

1,21

0,90

CI: Uper (g)

1,84

1,73

2,54

1,94

2,22

2,08

Skewness

0,32

0,27

0,10

0,30

0,38

0,28

Kurtosis

2,11

1,10

1,49

2,33

2,81

7,29

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

223

Table 31: Measure of central tendency and dispersion for Total power ms2 FFT [K] Experimental group Total power 2 ms FFT [K]

t0

t1

Sham group t2

t0

t1

t2

Mean

3090,69

3490,47

↑

2349,97

↓

2627,36

3404,74

↑

3283,49

↑

Median

1894,60

3186,25

↑

2290,36

↑

1677,22

1692,58

↑

2344,08

↑

70%

↑

50%

↓

65%

↑

65%

↑

* CVi

0,47

0,50

0,79

0,77

§

1,79

&

12,94

CVg

SD

1,29

3974,73

SEM (i)

2756,15

↓

0,25

0,32

1,23

1,00

63,91

1,33

56,36

-23,97

29,59

1802,89

348,86

303,28

616,29

403,14

CI: Lower (i)

2606,81

CI: Uper (i)

3974,35

SEM (g)

888,78

1,23

↓

3236,51

4172,08

24,97 ↑

3294,87

170,77

208,26

932,90

736,75

2125,91

2681,34

2547,23

3314,75

3350,76

3363,62

723,70

CI: Lower (g)

1348,69

2282,54

1559,82

1208,90

1576,24

1839,45

CI: Uper (g)

4832,69

4698,41

3140,12

4045,83

5233,23

4727,52

Skewness

0,30

0,11

0,03

0,29

0,41

0,29

Kurtosis

9,42

-0,07

-1,02

10,21

4,48

4,63

↑

Table 32: Measure of central tendency and dispersion for Total power ms2 AR [K] Total power 2 ms AR [K]

Experimental group t0

t1

Sham group t2

t0

Mean

3250,60

3574,74

↑

2285,09

Median

1886,45

2858,54

↑

2135,18

↓ ↑

65%

↑

60%

↓

* CVi CVg

1,29

0,43

0,51

0,71

0,76

t1

t2

2724,97

3689,84

↑

3456,98

↑

1841,17

2079,09

↑

2296,89

↑

75%

↑

70%

↑

1,15

0,24

0,30

1,24

1,04

§

1,23

41,23

1,04

42,19

&

9,97

-29,70

35,41

26,86

SD

4206,92

SEM (i) SEM (g)

940,70

CI: Lower (i) CI: Uper (i)

2549,86

↓

1740,26

326,72

313,73

570,17

389,13

2772,29

2152,92

↓

3137,76 701,62

4576,05

↑

3594,71

170,69

204,69

1023,24

803,80

2872,84

2689,79

4053,04

3382,76

3541,97

3492,17

CI: Lower (g)

1406,84

2457,21

1522,38

1349,79

1684,30

1881,53

CI: Uper (g)

5094,36

4692,26

3047,79

4100,16

5695,38

5032,43

Skewness

0,32

0,28

0,09

0,28

0,35

0,32

Kurtosis

11,13

-0,14

-1,00

8,28

5,01

3,87

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

224

14.9.3 HRV: Nonlinear analysis Table 33: Measure of central tendency and dispersion for ApEn ApEn

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

1,28

1,29

↑

1,33

↑

1,31

1,30

↓

1,33

↑

Median

1,28

1,30

↑

1,32

↑

1,33

1,32

↓

1,32

↓

60%

↑

70%

↑

55%

↓

50%

↓

* CVi

0,04

0,05

0,07

0,07

§

90,72

&

0,66

CVg

0,09

SD

0,12

SEM (i) SEM (g)

0,03

0,09

↓

0,02

0,04

0,09

0,06

4,85

-0,40

2,32

4,06

-0,09

1,68

0,10

0,01

0,01

0,09

↓

0,12

0,03

0,12

=

0,08

0,01

0,01

0,02

0,02

0,03

0,02

CI: Lower (i)

1,26

1,28

1,29

1,29

CI: Uper (i)

1,31

1,33

1,32

1,34

CI: Lower (g)

1,23

1,25

1,29

1,25

1,25

1,29

CI: Uper (g)

1,33

1,32

1,37

1,36

1,36

1,36

Skewness

0,00

-0,11

0,10

-0,17

0,17

0,13

Kurtosis

2,09

-0,47

1,58

0,28

-0,28

-0,43

↓

Table 34: Measure of central tendency and dispersion for SD1 SD1 (ms)

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

42,23

44,85

↑

29,29

↓

38,24

41,60

↑

45,72

↑

Median

32,38

39,02

↑

29,03

↓

30,78

31,03

↑

36,81

↑

90%

↑

60%

↓

95%

↑

75%

↑

* CVi

0,19

0,35

0,56

0,59

§

332,39

&

6,19

CVg

SD

0,81

34,02

SEM (i)

25,18

↓

0,08

0,15

0,68

0,63

-13,75

280,41

20,57

-30,65

8,78

19,56

17,33

1,88

2,81

5,63

3,88

CI: Lower (i)

39,85

CI: Uper (i)

47,23

SEM (g)

7,61

0,64

↓

24,63

28,10

↑

28,71

0,70

1,44

6,28

6,42

30,25

38,54

39,15

41,27

41,30

44,81

5,51

CI: Lower (g)

27,32

33,81

21,69

27,44

29,28

33,13

CI: Uper (g)

57,14

55,89

36,88

49,04

53,91

58,30

Skewness

0,29

0,23

0,02

0,30

0,38

0,31

Kurtosis

6,58

0,52

-0,22

1,28

2,76

-0,18

↑

225

Table 35: Measure of central tendency and dispersion for SD2 SD2 (ms)

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

60,15

68,01

↑

53,28

↓

57,50

66,74

↑

62,84

↑

Median

52,22

67,69

↑

51,62

↓

52,97

54,48

↑

58,52

↑

75%

↑

55%

↓

75%

↑

70%

↑

* CVi CVg

0,54

§ &

0,22

0,22

0,40

0,45

26,13

-2,40

13,05

SD

32,39

SEM (i)

27,14

-11,43 ↓

3,09

SEM (g)

7,24

0,45

24,24

0,12

0,16

0,54

0,42

18,96

13,42

16,07 ↓

25,72

2,73

36,35

9,29 ↑

1,73 5,75

26,09

↑

2,11

6,07

5,42

8,13

5,83

CI: Lower (i)

58,03

51,36

58,73

56,04

CI: Uper (i)

70,13

62,07

65,52

64,31

CI: Lower (g)

45,96

56,11

42,65

46,23

50,81

51,41

CI: Uper (g)

74,35

79,90

63,90

68,78

82,68

74,28

Skewness

0,24

0,01

0,68

0,18

0,34

0,17

Kurtosis

3,27

-0,77

-1,11

3,25

3,80

1,90

Table 36: Measure of central tendency and dispersion for D2 D2

Experimental group t0

Mean

2,94

Median

3,81

* CVi CVg

0,58

§ & SD

t1 3,34

↑

4,06 60%

↓

2,58

↑

2,87

↓

3,43

↑

60%

↑

0,14

0,27

0,49

0,80

2759,89

0,87

SEM (i)

1,62

1,83 0,16

0,36

0,41

CI: Lower (i)

2,94

CI: Uper (i)

3,33 2,63

1,48

CI: Lower (g)

0,38

2,18

0,59

-22,40 ↓

0,10

SEM (g)

t0

2,28

13,53 1,72

Sham group t2

t1

t2

2,63

↑

2,99

↑

2,93 60%

↓

3,48

↑

↓

60%

↑

0,19

0,18

0,60

0,53

741,65

36,39

2,01 ↑

1,52

1,59

16,21 ↑

1,58

0,11

0,11

0,36

0,35

2,30

2,38

2,57

2,92

2,82

3,00

1,93

2,30

0,34

1,91

CI: Uper (g)

3,69

4,04

3,08

3,24

3,32

3,68

Skewness

-0,51

-0,44

-0,32

-0,56

-0,19

-0,31

Kurtosis

-1,12

0,41

-1,96

-1,71

-1,69

-1,07

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

226

Table 37: Measure of central tendency and dispersion for DFA α1 Experimental group

Sham group

DFA α1 t0 Mean

0,95

Median

0,92

* CVi

t1

t2

0,98

↑

0,97 55%

1,06

↑

0,97

↑

1,06

↑

0,99

↑

70%

↑

0,11

0,17

0,22

0,20

§

529,60

&

2,50

CVg

0,28

SD

0,26

SEM (i) SEM (g)

0,06

CI: Lower (i) CI: Uper (i)

0,21

t0

↓

t1

t2

1,02

↑

1,00 60%

0,92

↓

↑

0,91

↓

↓

65%

↑

0,08

0,10

0,18

0,32

17,98

862,02

-4,21

11,56

5,52

-4,50

0,22

0,02

0,04

0,05

0,05

0,92

0,93

0,24

↓

0,24 0,05

0,18

↓

0,29

0,02

0,02

0,04

0,07

0,96

0,90

1,01

1,08

1,03

0,99

CI: Lower (g)

0,84

0,88

0,97

0,86

0,94

0,80

CI: Uper (g)

1,07

1,07

1,16

1,07

1,10

1,05

Skewness

0,12

0,05

0,00

-0,08

0,11

0,03

Kurtosis

-1,21

0,34

-1,42

-0,40

-0,97

-0,02

↑

Table 38: Measure of central tendency and dispersion for DFA α2 Experimental group

DFA α2 t0 Mean Median

0,25 0,23

* CVi

t1

Sham group t2

↑ ↑

0,37 0,37

↑ ↑

55%

↑

70%

↑

0,25

0,29

0,51

0,31

§

8467,17

&

30,06

CVg

SD

0,37

0,09

SEM (i) SEM (g)

0,02

t0

0,32 0,29

0,16

↑

0,26 0,25

t1

t2

0,29 0,29

↑ ↑

0,27 0,23

↑ ↓

65%

↑

55%

↓

0,15

0,16

0,39

0,44

60,37

7505,01

8,41

49,23

13,59

3,44

0,11

0,02

0,02

0,42

↑

0,11

0,02

0,11

=

0,12

0,01

0,01

0,04

0,03

0,03

0,03

CI: Lower (i)

0,25

0,27

0,26

0,24

CI: Uper (i)

0,31

0,34

0,29

0,28

CI: Lower (g)

0,21

0,25

0,32

0,21

0,24

0,21

CI: Uper (g)

0,29

0,39

0,41

0,30

0,34

0,32

Skewness

0,22

0,19

0,00

0,09

0,00

0,33

Kurtosis

0,95

1,97

-1,03

-0,74

0,81

0,07

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

227

Tabel 39: Measure of central tendency and dispersion for Shannon Entropy Shannon Entropy

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

3,07

3,10

↑

3,11

↑

3,02

3,11

↑

2,99

↓

Median

2,97

3,04

↑

3,06

↑

3,02

3,08

↑

3,03

↑

50%

↑

70%

↑

60%

↓

*

65%

CVi

0,05

0,05

0,08

0,07

§

44,58

&

1,11

CVg

SD

0,10

0,31

SEM (i) SEM (g)

0,07

CI: Lower (i) CI: Uper (i)

0,26

↓

0,04

0,06

0,08

0,09

1,92

93,07

-0,43

1,19

2,96

-0,89

0,21

0,04

0,04

0,06

0,05

3,02

3,02

0,08

↓

0,24 0,05

0,26

↑

0,26

0,03

0,04

0,06

0,06

3,01

2,93

3,16

3,16

3,12

3,08

CI: Lower (g)

2,93

2,99

3,01

2,91

2,99

2,88

CI: Uper (g)

3,20

3,22

3,20

3,13

3,22

3,11

Skewness

0,32

0,23

0,24

0,00

0,12

-0,15

Kurtosis

4,04

3,59

-1,12

1,31

-0,02

-0,53

↑

228

14.9.4 Other measurements: SCL, PEP, RSA, respiration rate, blood pressure Table 40: Measure of central tendency and dispersion for SCL Experimental group

SCL (µS) t0 Mean

4,26

Median

3,13

* CVi

t1

Sham group t2

5,23

↑

4,40 75%

5,05

↑

3,26

↑

4,42

↑

3,49

↑

75%

↑

0,22

0,34

0,61

0,72

§

782,28

&

22,90

CVg

0,80

SD

3,42

SEM (i) SEM (g)

0,76

CI: Lower (i) CI: Uper (i)

3,17

t0

↓

t1

t2

4,75

↑

3,86

↑

4,66 85%

↑

3,12

↓

↑

50%

↓

0,27

0,34

0,50

0,74

32,82

1095,42

23,81

18,55

45,74

18,34

3,62

0,23

0,35

0,71

0,81

4,29

3,97

5,20

5,34

CI: Lower (g)

2,76

3,84

3,46

CI: Uper (g)

5,76

6,62

Skewness

0,33

0,26

Kurtosis

4,08

-0,41

0,40

↑

1,29 0,29

2,37

↑

2,85

0,25

0.27

0,53

0,64

3,53

3,03

4,49

4,09

2,70

3,71

2,61

6,63

3,83

5,79

5,11

0,17

-0,18

0,04

0,26

2,56

-0,03

-0,26

0,66

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intra-individual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

Table 41: Measure of central tendency and dispersion for PEP PEP (ms)

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

90,64

88,43

↓

94,59

↑

90,55

94,56

↑

91,76

↑

Median * CVi

86,46

88,87 60% 0,05

↑ ↑

99,44 65% 0,07

↑ ↑

92,81

97,73 65% 0,05

↑ ↑

91,18 70% 0,04

↓ ↑

CVg

0,22

0,18

0,23

0,16

0,14

0,15

§

-5,60

6,25

6,12

2,36

&

-2,43

4,36

4,43

1,34

SD

19,71

SEM (i) SEM (g)

4,41

CI: Lower (i) CI: Uper (i)

20,08

↑

15,02

1,00

1,54

4,49

3,36

87,58

89,59

↓

16,24 3,63

13,32

↓

13,87

0,96

0,92

2,98

3,10

90,68

89,36

91,48

95,63

94,43

92,95

CI: Lower (g)

82,00

79,63

88,01

83,43

88,72

85,68

CI: Uper (g)

99,27

97,23

101,17

97,67

100,39

97,84

Skewness

0,21

-0,02

-0,32

-0,14

-0,24

0,04

Kurtosis

0,20

0,14

-1,19

-0,69

0,30

-1,27

↓

229

Table 42: Measure of central tendency and dispersion for RSA RSA

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

82,22

91,71

↑

65,72

↓

75,18

79,23

↑

82,80

↑

Median

74,68

91,50

↑

54,40

↓

71,47

68,29

↓

67,30

↓

65%

↑

70%

↓

60%

↑

75%

↑

* CVi

0,19

0,27

0,49

0,59

§

17,93

&

11,54

CVg

SD

0,53

43,67

SEM (i)

45,21

↑

0,09

0,20

0,49

0,57

-13,11

3,96

10,53

-20,07

5,39

10,13

38,99

3,60

4,42

10,11

8,72

CI: Lower (i)

79,91

CI: Uper (i)

94,03

SEM (g)

9,77

0,45

↓

33,55

38,62

↑

47,00

1,47

3,48

8,64

10,51

65,31

74,32

72,18

82,63

80,09

85,80

7,50

CI: Lower (g)

63,08

71,90

48,63

60,48

62,31

62,20

CI: Uper (g)

101,37

111,53

82,81

89,88

96,16

103,39

Skewness

0,17

0,00

0,29

0,11

0,28

0,33

Kurtosis

-0,43

-0,94

-0,86

0,35

0,36

0,60

↑

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intraindividual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

Table 43: Measure of central tendency and dispersion for respiration rate Respi. rate (bpm)

Experimental group t0

t1

Sham group t2

t0

t1

t2

Mean

15,81

15,63

↓

16,61

↑

16,00

16,02

↑

16,70

↑

Median

15,30

15,68

↑

16,82

↑

16,07

15,17

↓

17,14

↑

65%

↓

65%

↑

55%

↓

55%

↑

* CVi CVg

0,18

§ & SD

0,09

0,17

0,18

-5,94

6,14

-1,17 2,85

SEM (i) SEM (g)

0,05

2,68 0,19

0,64

0,17

5,06 ↓

2,95

0,05

0,06

0,16

0,14

3,84

5,68

0,12 ↑

2,70

0,32

2,49 0,18

0,60

4,40 ↓

2,40

↓

0,23

0,60

0,66

0,56

0,54

CI: Lower (i)

15,35

15,59

15,66

15,90

CI: Uper (i)

16,09

16,83

16,36

16,80

CI: Lower (g)

14,56

14,45

15,32

14,82

14,93

15,65

CI: Uper (g)

17,06

16,80

17,90

17,18

17,11

17,76

Skewness

0,18

-0,02

-0,07

-0,03

0,34

-0,18

Kurtosis

-0,05

-0,46

-0,84

-1,22

0,32

-1,27

230

Table 44: Measure of central tendency and dispersion for SBPR SBPR (mm Hg)

Experimental group t0

t1

t3

Sham group t2

t0

t1

t3

t2

Mean

117,00

117,10

↑

114,20 ↓

113,05

↓

120,95

119,15

↓

114,15 ↓

113,40

↓

Median

115,50

117,00

↑

113,50 ↓

113,00

↓

115,50

111,50

↓

115,50

113,50

↓

65%

↑

60%

65%

↓

75%

↓

70%

75%

↓

* CVi

↓

0,04

0,03

0,05

0,12

0,10

0,11

§

-0,36

-2,22

&

0,09

-2,39

CVg

SD SEM (i) SEM (g) CI: Lower (i) CI: Uper (i) CI: Lower (g) CI: Uper (g)

0,10

11,68

14,56

↑

11,15 ↓

0,05

0,06

0,06

0,16

0,09

0,09

-3,17

-1,66

-4,79

-4,82

-3,38

-1,49

-5,62

-6,24

12,04

111,88

0,95 3,26 115,19 118,91 110,72

0,87 2,49 113,90 117,90 109,31

1,19 2,69 112,69 117,36 107,77

122,12

123,48

119,09

118,33

2,61

= ↓

0,14

↑

17,47

18,85

↑

10,07 ↓

10,14

113,29

1,30 4,21 117,51 122,59 110,89

1,58 2,25 114,45 120,65 109,74

1,54 2,27 114,15 120,20 108,95

128,61

127,41

118,56

117,85

3,91

Skewness

0,13

0,01

0,06

0,00

0,31

0,41

-1,35

-0,01

Kurtosis

-0,90

-0,31

-0,21

0,01

5,45

2,60

0,11

2,13

↓

*: inter-individual differences between measures (increase or decrease) compared between the group; CVi: intraindividual(i) coefficient of variations ; CVg: inter-individual(g) coefficient of variations; §: Average actual change within-subjects; &: Average actual change between-subjects; SD: Standard deviation; SEM: Standard Error of the mean; CI: confidence interval 95%

Table 45: Measure of central tendency and dispersion for DBPR DBPR (mm Hg)

Experimental group t0

t1

t3

Sham group t2

t0

t1

t3

t2

Mean

74,05

75,10

↑

71,35 ↓

71,60

↓

79,10

77,70

↓

75,80 ↓

76,20

↓

Median

75,50

73,50

↓

71,00 ↓

72,00

↓

76,50

73,50

↓

73,50 ↓

72,00

↓

55%

↑

70%

↓

60%

↓

50%

↓

60%

↓

65%

↓

* CVi CVg

0,10

§ & SD

0,06

0,05

0,13

0,11

0,12

1,28

-3,10

-5,14

1,42 7,63

SEM (i) SEM (g)

0,06

9,97 1,00

1,71

3,65 ↑

8,03 ↑ 1,01

0,13

-3,31 8,70

0,05

0,05

0,05

0,13

0,10

0,13

-2,19

-3,52

-4,69

-1,77 ↑

10,59

0,86

9,96 0,84

2,37

-4,17 ↓

7,67 ↓ 0,87

-3,67 ↓

9,60 0,93

2,23

1,80

1,94

2,23

1,72

2,15

CI: Lower (i)

72,61

70,72

71,14

76,76

75,75

75,82

CI: Uper (i)

76,54

74,68

74,51

80,04

79,15

79,48

CI: Lower (g)

70,71

70,73

67,83

67,79

74,46

73,33

72,44

71,99

CI: Uper (g)

77,39

79,47

74,87

75,41

83,74

82,07

79,16

80,41

Skewness

-0,19

0,16

0,04

-0,45

0,25

0,42

0,30

0,44

Kurtosis

0,11

1,29

-0,14

-1,43

0,51

0,09

1,61

-0,59

231

14.9.5 Regression to the mean Table 46: Regression to the mean Experimental group Variable t2

Regression to the mean (RTM) Total

Number RTM

% RTM

LF n.u. AR [K]

15

4

26,67

2

HF ms FFT [V]

13

1

7,69

HF n.u. AR [K] 2 LF/HF ratio ms FFT [V] DFA α2

14

3

21,43

15

2

13,33

19

5

26,32

232

14.10 Addendum 10: Techniques used in the experimental group 1. Third Vault Hold Therapist: sits on a chair at the head end of the treatment table with the feet supported on the ground. Both elbows are supported on the knees. Take up a position at the head of the patient. Bring the head of the subject in to neutral position. Bring the head of the subject into his/ her preferred position (follow the easy way).

Height of the treatment table: the treatment table is at knee height of the seated therapist.

Position of the subject: subject lies supine on the treatment table, the legs are extended and the arms lie next to the body. The head is supported by a pillow.

Hand position: place your thumbs on the great wings of the sphenoid, behind the lateral cornes of the eyes. Your index fingers should be positioned on the temporal bones anterior to the occipitomastoid suture. Position your middle, ring and little fingers on the occiput.

2. Spheno- basilar decompression: ('Platybasia technique') Therapist: sits on a chair at the head end of the treatment table with the feet supported on the ground. Both elbows are supported on the knees. Take up a position at the head of the patient. Bring the head of the subject in to neutral position. Bring the head of the subject into his preferred position (follow the easy way).

Height of the treatment table: the treatment table is at knee height of the seated therapist.

Position of the subject: subject lies supine on the treatment table, the legs are extended and the arms lie next to the body. The head is supported by a pillow.

Hand position: place your thumbs on the greater wings of the sphenoid. Your index fingers should be positioned on the temporal bones anterior to the occipitomastoid suture. Position your middle, ring and little fingers on the occiput.

Method: Anterior-posterior decompression of the sphenobasilar synchondrosis. With your thumbs on the greater wings of the sphenoid, apply traction in an anterior direction.

3. J. E. Upledger’s ‘ear pull’ technique Therapist: sits on a chair at the head end of the treatment table with the feet supported on the ground. Both elbows are supported on the knees. Take up a position at the head of the patient. Bring the head of the subject in to neutral position. Bring the head of the subject into his preferred position (follow the easy way).

Height of the treatment table: the treatment table is at knee height of the seated therapist.

Position of the subject: subject lies supine on the treatment table, the legs are extended and the arms lie next to the body. The head is supported by a pillow.

233

Hand position: Place your thumbs in the external acoustic meatus of each ear. Place your index and middle fingers behind the ear lobes, as close as possible to the temporal bones.

Grasp the antitragus and lobe of each ear with your thumb and fingers.

Method: apply traction in a lateroposterior superior direction and at an angle, roughly in a continuous line with the petrous parts. Gently, very slowly increase the amount of force. A slightly greater application of force can sometimes be needed to release restrictions of the temporal bone, but this must always be kept below the point where the tissue reacts to the force of the traction and contracts against it. While administering the traction you can make constant slight adjustments to the angle of pull to correspond to the tension pattern of the tentorium and the sutures. Permit all motions/tissue unwinding of the tentorium by following the motions passively with your hands, without reducing the gentle traction. This traction first releases the sutural joints between the petrous part and the corresponding joint surfaces of the sphenoid and occiput, followed by the various membranous tensions of the tentorium (osseous release, elastic/collagen membrane and fluid release). When the tentorium has been released form its tension patterns, you can gradually relax the lateroposterior superior traction and then remove your hands.

Important: When performing the release technique, never remove your hands suddenly from the bone. Doing so can cause dysfunctions.

4. Parietal lift Therapist: sits on a chair at the head end of the treatment table with the feet supported on the ground. Both elbows are supported on the knees. Take up a position at the head of the patient. Bring the head of the subject into neutral position. Bring the head of the subject into his preferred position (follow the easy way).

Height of the treatment table: the treatment table is at knee height of the seated therapist.

Position of the subject: subject lies supine on the treatment table, the legs are extended and the arms lie next to the body. The head is supported by a pillow.

Hand position: place the palms of both hands on the parietal tubers. The tips of your thumbs should touch each other at the sagital but without resting on the suture. Position your middle fingers above the roots of the zygomatic processes of the temporal bones. Position your ring fingers on the mastoid angles (posterior inferior angles of the parietal bones). Place the basal or proximal joints of your index, middle and ring fingers on the parietal bone.

Position them on the lateral borders of the parietal bones.

Method: during the expiration phase, begin to administer gentle medial pressure with your index, middle and ring fingers on the inferior lateral borders of the parietal bones, to release them from the temporal bones. As soon the parietal bones begin to move cephalad (release of the parietal bones from the temporal bones), relax the medial pressure of your fingers.

Replace this with cephalad traction with your fingers. At the same time, without reducing the cephalad traction, you can administer slight posterior traction to release the parietal bones from the frontal bone, and then slight anterior traction to release the parietal bones from the occipital bone. Very gently the parietal bones are moved out of the field of tension. The therapist stays within the range of motion, meaning that he avoids confronting the motion barriers directly. The tissue dynamics are just perceived without intervention. You will be able to sense the various stages of tissue release: First the sutural tensions, then the elastic and collagenous tensions of the falx cerebri. In the end a new equilibrium of tension will establish itself and a floating sensation may be perceived.

234

5. Frontal lift Therapist: sits on a chair at the head end of the treatment table with the feet supported on the ground. Both elbows are supported on the knees. Take up a position at the head of the patient. Bring the head of the subject into neutral position. Bring the head of the subject into his preferred position (follow the easy way).

Height of the treatment table: the treatment table is at knee height of the seated therapist.

Position of the subject: lies supine on the treatment table, the legs are extended and the arms lie next to the body. The head Is supported by a pillow.

Hand position: place your ring fingers on the outside of the zygomatic processes of the frontal bone. Grasp these securely from outside with your inbent fingers, to provide a firm hold. Place your little fingers beside your ring fingers. Position your middle and index fingers laterally adjacent to the midline of the frontal bone. Your thumbs should lie posteriorly, either crossed or touching.

Method: during the expiration phase, begin to apply gentle pressure in a medial direction with the ring fingers on the lateral surface of the frontal bone so as to release it from the sphenoid. As soon as the frontal bone begins to move anteriorly you can relax the medial pressure of your ring fingers. Replace this pressure with an anterior, slightly cephalad traction. Induce the anterior traction by pressing your elbows slightly down into the table, so that your fingers lift anterior. Very gently the frontal bone is moved out of the field of tension.

The therapist stays within the range of motion, meaning that he avoids confronting the motion barriers directly. The weight of the skull is enough to hold the occiput, the posterior position on the table. The tissue dynamics are just perceived without intervention.

6. Technique for the frontonasal suture Therapist: sits on a chair at the head end of the treatment table with the feet supported on the ground. The right elbow is supported on the treatment table. Take up a position at the head of the patient. Bring the head of the subject into neutral position. Bring the head of the subject into his preferred position (follow the easy way).

Height of the treatment table: the treatment table is at knee height of the seated therapist.

Position of the subject: lies supine on the treatment table, the legs are extended and the arms lie next to the body. The head is supported by a pillow.

Hand position: Cranial hand: - Place your cranial hand on the frontal bone with your middle finger on the metopic suture, immediately above the frontonasal suture.

- Place your index and ring fingers next to the middle finger.

Caudal hand: - Place your thumb and index finger one on each nasal bones.

Method: register the cranial movements of the frontal bone with your hand resting passively on the frontal bone. During the inspiration phase, deliver an impulse to external rotation with the thumb and index finger that are on the nasal bones. During the expiration phase, deliver

235

an impulse into internal rotation with the thumband index finger that are on the nasal bones.

Administer gentle traction cephalad on the frontal bone. At the same time administer traction caudad on the nasal bones.

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15 Acknowledgements Thanks to all the team members and ‘VZW Health’ for their -mostly indirect- help in the design of this study. Thanks to Janssens P. for the external control of the FMEA. Thanks to Flanders International College of Osteopathy for the logistic support of the building.

Thanks to Vydt J., El Mokkari S., Janssens P. with their support of the English translation.

Thanks to Van Aert S. for the lay-out of this dissertation.

Special thanks go to Smeets H. for the realization of the RILHOU-RCT and De Cock JJ. († 6/12/2016) that act as scientific coordinator and had each time the patience and refined the last points.

Without the support and the belief in me as a person of all the people above and especially my surroundings (son, girlfriend, parents, family, friends and colleagues), without these wonderful people, it was impossible to obtain my Diploma in Osteopathy.

“From the bottom of my heart, thank you all so much!

It really means a lot to me!”

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