The Impact of Intermittent Fasting on Human Metabolism and Cell Autophagy
Phase: NA
Conditions: Excessive Diet Restriction
Sponsor: Medical University of Graz

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 1 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
Intermittent Fasting Cohort Study 
“InterFast” 
Ethics number: 27-166 ex 14/15 
Protocol code:  HS – 2014 – 03  
Date and version number:  01/06/2018                 Version 1.9 
Registration with ClinicalTrials.gov:  NCT02673515 
Chief Investigator: 
Univ.-Prof. Dr. Frank Madeo 
Univ.-Prof. Dr. Thomas Pieber  
Principal Investigator: 
Assoz.-Prof. Dr. Harald Sourij  
Project Manager: 
Norbert Tripolt, PhD 
Slaven Stekovic, BSc. MSc.  
Co-Investigators: 
Univ.-Prof. Dr. Barbara Obermayer-Pietsch 
Dr. Anja Ribitsch 
Dr. rer. nat. Tobias Eisenberg 
Dr. rer. nat. Sabrina Schröder 
Filomena Broeskamp, BSc. 
Biometrician: 
MMag. Sophie Narath 
Trial site address and telephone number: 
Medical University of Graz, Department of Endocrinology and Diabetology, Cardiovascular Diabetology Research 
Group, Austria, 8036 Graz, Auenbruggerplatz 15; Telephone: +43 (0)316 385 13270 Fax: +43 (0)316 385 14332 
Signatures: 
 
Univ.-Prof. Dr. Frank Madeo:    _________________________________________________ 
 
Univ.-Prof. Dr. Thomas Pieber: _________________________________________________ 
               Assoz.-Prof. Dr. Harald Sourij:  _________________________________________________ 
 
CONFIDENTIAL 
This document contains confidential information that must not be disclosed to anyone other than the sponsor, the 
investigator team, regulatory authorities and members of the Research Ethics Committee. 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 2 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
1. 
TABLE OF CONTENTS 
1. 
TABLE OF CONTENTS................................................................................................................................... 2 
2. 
LIST OF ABBREVIATIONS .......................................................................................................................... 557 
3. 
AMENDMENT HISTORY ............................................................................................................................ 668 
4. 
SYNOPSIS ................................................................................................................................................. 668 
5. 
BACKGROUND AND RATIONALE .............................................................................................................. 779 
6. 
AIMS OF THE STUDY ................................................................................................................................ 779 
7. 
OBJECTIVES ............................................................................................................................................ 8810 
7.1 
PRIMARY OBJECTIVE ..................................................................................................................................... 8810 
7.2 
SECONDARY OBJECTIVE ................................................................................................................................. 8810 
7.3        LONG-TERM OBJECTIVE ................................................................................................................................ 8810 
8. 
OUTCOMES ............................................................................................................................................ 8810 
8.1 
PRIMARY OUTCOME MEASURES IN THE COHORT STUDY ........................................................................................ 8810 
8.2 
SECONDARY OUTCOME MEASURES IN THE COHORT STUDY .................................................................................... 8810 
8.3      PRIMARY OUTCOME MEASURES IN THE RANDOMIZED CONTROLLED TRIAL ................................................................ 9911 
8.4      SECONDARY OUTCOME MEASURES IN THE RANDOMIZED CONTROLLED TRIAL ............................................................ 9911 
9.    STUDY DESIGN ..................................................................................................................................... 101012 
9.1   SCHEMATIC OVERVIEW ON STUDY DESIGN ....................................................................................................... 101012 
9.2           DURATION OF STUDY ............................................................................................................................ 101012 
9.3         DATA COLLECTION .................................................................................................................................. 101012 
9.4        RANDOMIZATION .................................................................................................................................... 111113 
10.    SELECTION AND WITHDRAWAL OF STUDY SUBJECTS ......................................................................... 111113 
10.1   SUBJECT SELECTION .................................................................................................................................. 111113 
10.3       INCLUSION CRITERIA .............................................................................................................................. 111113 
10.4     EXCLUSION CRITERIA ............................................................................................................................... 111113 
10.5     WITHDRAWAL / DROP OUT OF SUBJECTS ..................................................................................................... 121214 
10.6      PAYMENT TO SUBJECTS ............................................................................................................................ 121214 
11.    STUDY PROCEDURES .......................................................................................................................... 121214 
11.1  SCHEDULE OF STUDY PROCEDURES OF THE COHORT STUDY ................................................................................ 121214 
11.2   VISIT SCHEDULE FOR THE COHORT STUDY ...................................................................................................... 131315 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 3 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
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Tel: ++43 (0) 316 385 13270
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
11.3  SCHEDULE OF STUDY PROCEDURES OF THE PILOT INTERVENTION TRIAL ................................................................. 161618 
11.4   LABORATORY RESULTS ............................................................................................................................... 161618 
12.   METHODS ........................................................................................................................................... 171719 
12.1   ORAL GLUCOSE TOLERANCE TEST (OGTT) ..................................................................................................... 171719 
12.2   ENDOTHELIAL FUNCTION ............................................................................................................................ 171719 
12.3   BONE DENSITOMETRY AND BODY COMPOSITION ............................................................................................. 181820 
12.4   LABORATORY MEASUREMENTS (CLINICAL AND HORMONAL) .............................................................................. 181820 
12.5   LABORATORY MEASUREMENTS (SPECIALIZED RESEARCH – AGING PARAMETERS) .................................................. 191921 
12.6   PHYSICAL ACTIVITY MEASUREMENT .............................................................................................................. 191921 
12.7   INTIMA MEDIA THICKNESS (IMT) ................................................................................................................. 202022 
12.8   FOOD FREQUENCY QUESTIONNAIRE (FFQ) AND HORMONAL SCORES................................................................... 202022 
12.9   NON-INVASIVE 24H AMBULATORY BLOOD PRESSURE MONITORING .................................................................... 202022 
12.10    RESTING ENERGY EXPENDITURE (REE) ....................................................................................................... 212123 
12.11   HAND GRIP TEST .................................................................................................................................... 212123 
12.12   PULSE WAVE ANALYSIS ............................................................................................................................ 212123 
12.13   ECHOCARDIOGRAM (ECG) ....................................................................................................................... 222224 
12.14   BUCCAL MUCOSA SAMPLING ..................................................................................................................... 222224 
12.15   BLOOD SAMPLING AND STORAGE ............................................................................................................... 222224 
12.16   FAECES SAMPLING .................................................................................................................................. 232325 
12.17   SALIVA SAMPLING ................................................................................................................................... 232325 
12.18  CONTINUOUS GLUCOSE MONITORING ......................................................................................................... 232325 
12.20  VITAL SIGNS ........................................................................................................................................... 242426 
12.21  ECHOCARDIOGRAPHY ............................................................................................................................... 242426 
12.22   INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE (IPAQ) .......................................................................... 242426 
12.23   BIOELECTRICAL IMPEDANCE ANALYSIS (BIA) ................................................................................................ 252527 
12.25   FLOW MEDIATED DILATATION (FMD) ......................................................................................................... 262628 
12.26   DATA ANALYSIS AND SAMPLE SIZE ESTIMATION ............................................................................................. 262628 
Data analysis ............................................................................................................................................ 262628 
Sample size estimation ............................................................................................................................. 272729 
12.27   MARKERS OF CARDIOVASCULAR RISK .......................................................................................................... 272729 
13.    STUDY MONITORING ......................................................................................................................... 272729 
14.    ETHICS ............................................................................................................................................... 282830 
14.1   INFORMED CONSENT ................................................................................................................................ 282830 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 4 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
14.2    ETHICAL AND REGULATORY APPROVALS ....................................................................................................... 292931 
15.    FINANCE ............................................................................................................................................ 292931 
16.    INSURANCE ........................................................................................................................................ 292931 
17.    REFERENCES ....................................................................................................................................... 303032 
 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 5 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
2. 
LIST OF ABBREVIATIONS 
 
AIx  Aortic augmentation index 
BMCyt  Buccal Micronucleus Cytome 
DBP  diastolic blood pressure 
DEGS  German Health Examination Survey for Adults 
DEXA  dual-energy x-ray absorptiometry 
ECG  Echocardiogram 
EDTA  ethylenediaminetetraacetic 
FFQ  Food frequency questionnaire 
FMD  Flow mediated dilatation 
FPFV  First patient  first visit 
IC  indirect calorimetry 
IMT  Intima media thickness 
IPAQ  International physical activity questionnaire 
LPFV  Last patient first visit 
LPLV  Last patient last visit 
MN  micronucleus 
NaCl  Sodium Chloride 
NEMONIT  German National Nutrition Monitoring 
OGTT  oral glucose tolerance test 
PAT  peripheral arterial tone 
PWA  Pulse wave analysis 
PWV  pulse wave velocity 
RCT  Randomised controlled trial 
REE  resting energy expenditure 
RHI  reactive hyperemia index 
SBP  systolic blood pressure 
Tris HCl  Tris hydrochloride 
 
 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 6 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
3. 
AMENDMENT HISTORY 
Amendment 
No. 
Protocol 
Version No. 
Date issued 
Author(s) of 
changes 
Details of Changes made 
1 
1.2 
10.04.2015 
Sourij, Tripolt 
Glucose monitoring device was 
added (MedTronic Ipro 2) 
2 
1.3 
13.05.2015 
Sourij, Tripolt 
Bioelectrical impedance analysis 
(BIA) was added 
3 
1.4 
03.07.2015 
Sourij, Tripolt 
Dry eye assessment, Ocular 
Surface 
Disease 
Index 
Questionnaire 
4 
1.5 
22.09.2015 
Sourij, Tripolt 
Changes in inclusion criteria 
(body 
mass 
index:22.0-
30.0kg/m2) 
5 
1.6 
10.02.2016 
Sourij, Tripolt 
FMD (Flow Mediated Dilatation) 
was added 
6 
1.7 
13.03.2016 
Sourij, Tripolt 
Number of patients was reduced 
from 100 to 90. 
 
4. 
SYNOPSIS 
Study title 
InterFast 
Study design 
Cohort study with an embedded randomized controlled pilot trial 
Study participants 
Healthy subjects and subjects who do already practice Alternate Day 
Fasting 
Number of participants 
90 (30 Participants in Alternate Day Fasting group and 60 participants in the 
control group). Those participants in the control group will be asked to 
participate in a short randomized controlled trial, where they will be either 
allocated to an Alternative Day Fasting group or another control visit.  
Planned duration 
18 months 
Primary objective 
The primary objective is to elucidate in which extent alternate day fasting 
influences human physiology in healthy individuals in both, the short and 
long term. Data of the pilot RCT will help to design a larger trial investigating 
Alternate Day Fasting in various groups of interest. 
Secondary objectives 
The secondary objective of this study is to define novel molecular markers 
of aging and age-related diseases. 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 7 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
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Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
5. BACKGROUND AND RATIONALE 
Intermittent fasting is a dietary regimen defined by alternating fasting and “feeding” cycles. In addition 
to caloric restriction (a dietary regimen limited to a daily food intake lower than one’s daily caloric needs) 
only, intermittent fasting seems to activate cell autophagy (cellular “recycling” program) which 
potentially increases cellular stress resistance and removes accumulated molecules that are potentially 
toxic1-3. In fact, mice maintained on intermittent fasting without decreased overall food intake show 
effects on body weight reduction that equal and in some cases even exceed those of calorie restriction4, 
5. However, additionally, intermittent fasting combined with even a high-fat diet in the feeding periods 
protects mice from obesity, hyperinsulinemia, hepatic steatosis, and inﬂammation compared to controls 
that are fed an ad libitum high-fat diet despite the same calorie intake6, making this intermittent fasting 
regimen a promising approach to reduce morbidity and mortality in various species. 
The best described and most widely practiced version of intermittent fasting is the “alternate day diet” 
or “alternate day fasting” (ADF). In animal models, ADF consists of an ad libitum “feed day” alternated 
with a 100% restriction “fast day”. However in humans, this is often modified to allow a small amount of 
food consumption on the “fast day” (e.g. 25% of the individual´s energy needs). Findings from recent 
modified ADF studies showed 4-8% reduction in body weight after 8-12 weeks7-10. This reduction in body 
weight is usually escorted by reductions in triglycerides8, 10, 11, low density lipoprotein (LDL) cholesterol8, 
10, 11, systolic blood pressure10, insulin resistance7 and increases in LDL particle size7, 12, 13.  
However, knowledge about the molecular effects of the alternate day diet on human metabolism or 
autophagy is still scarce since detailed analyses of molecular and metabolic parameters remain 
unexplored, especially in healthy individuals. The overarching aim of this research project is to elucidate 
in which extent alternate day fasting (and thereby intermittent fasting) influences human physiology in 
healthy individuals in both short and long term. The secondary objective of this study is to define novel 
molecular markers of aging and age-related diseases. 
6. AIMS OF THE STUDY 
The overarching aim of our cohort study with embedded pilot randomized control trial is to 
investigate the effects of repeating fasting periods on human physiology, aging process and 
molecular cellular processes in humans. We will be able to study long term effects (subjects in the 
cohort study, who already practise ADF for a defined time period) and short term effects (subjects 
randomized to the ADF group) of this nutritional intervention. 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 8 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
7. OBJECTIVES 
7.1 
Primary objective 
The primary objective is to elucidate in which extent ADF (and thereby intermittent fasting) 
influences human physiology in healthy individuals in both short and long term.   
7.2 
Secondary objective 
The secondary objective of this study is to investigate novel molecular markers and cellular 
pathways of aging and age-related diseases and whether they are influenced by ADF.  
7.3        Long-term objective 
The information gained in this project will be used to develop a strategy for follow-up studies in 
model organisms, such as yeast, worms, fruit flies, mice and humans. As a long term goal, we 
want to identify new targets (metabolites, macromolecules and pathways) that can be used for 
more precise clinical diagnosis and in the treatment and prevention of aging and age-related 
diseases. We will accomplish this by mimicking such nutritional regimen in animal models 
(methods are already well established in Prof. Madeo’s or his collaborator’s laboratories) in 
combination with knock-out and knock-down experiments, that will help differentiate between 
correlative and causal connection between nutrition, health and disease.  
8. OUTCOMES 
8.1 
Primary outcome measures in the cohort study 
- Differences in body composition (fat mass and lean body mass) between groups 
- Differences in resting energy expenditure between groups 
- Differences in insulin sensitivity (Matsuda Index, ISI, HOMA-Index, QUICKI) between groups 
- Difference in glycaemic pattern (continuous glucose measurement) between the two groups 
- Differences in endothelial function (Mean Intima media thickness, Reactive Hyperaemia 
Index, Stiffness Index, Aortic augmentation index) between groups 
- Differences in mean 24h blood pressure between groups 
- Differences in hormonal regulation (fertility, appetite hormones and others) 
8.2 
Secondary outcome measures in the cohort study 
- 
Levels of different cell signaling and cell cycle proteins (p16, p21, p53, NCX, mTOR, 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 9 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
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Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
Ras, Rb) 
- 
Differences in parameters of cell death (apoptosis, stress resistance, mitochondrial 
damage) between groups 
- 
Differences in telomere length, double strand breaks, chromosomal aberrations, 
heterochromatin between groups 
- 
Differences in telomerase activity between groups 
- 
Differences in endocrine parameters of fertility and nutritional regulation 
8.3      Primary outcome measures in the randomized controlled trial 
- 
Difference in the changes of insulin sensitivity (Matsuda index, ISI, HOMA-Index, 
QUICKI) 
- 
Difference in the change of endothelial function 
8.4      Secondary outcome measures in the randomized controlled trial 
- 
Levels of different cell signalling and cell cycle proteins (p16, p21, p53, NCX, mTOR, Ras, 
Rb) 
- 
Differences in parameters of cell death (apoptosis, stress resistance, mitochondrial 
damage) between groups 
- Differences in telomere length, double strand breaks, chromosomal aberrations, 
heterochromatin between groups 
- Differences in telomerase activity between groups 
 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 10 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
9.    STUDY DESIGN 
9.1   Schematic overview on study design 
 
9.2           Duration of Study 
It is anticipated that the study will run for 15 months (from first patient first visit until last 
patient last visit). 
 
First patient  first visit (FPFV): 
May 2015 
Last patient first visit (LPFV): 
July 2016 
Last patient last visit (LPLV): 
August 2016 
Data base lock: 
September 2016 
First results: 
October 2016 
9.3         Data collection 
Once informed consent has been obtained, recruiting clinicians complete a CRF for each 
participant to record socio-demographic information, information about medical history and 
medications and dietary habits. Source documents comprise the CRF and hospital records, 
laboratory records and correspondence. All documents will be stored safely in a confidential 
manner.  The subject will be referred to by a unique study subject number/code, their initials 
and date of birth on all study-specific documentations. The only exceptions will be the signed 
Consent Forms, Subject Identification log and subject clinical file, all of which will be stored 
securely by the clinical site.  Source data will be made available for internal and external audits 
or inspections by regulatory authorities to authorised personnel only. 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 11 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
9.4        Randomization 
Randomization for the pilot RCT will be designed and performed by the “Randomizer tool” 
provided by the Institute of Medical Informatics, Statistics and Documentation of Medical 
University Graz, Austria.  
 
10.    SELECTION AND WITHDRAWAL OF STUDY SUBJECTS 
10.1   Subject Selection 
The study population will consist of 100 healthy subjects. Fifty participants of them should already 
practice Alternate Day Fasting. Subjects in the ADF and control group will at least be matched by 
sex, weight and age. Subjects of the control group will be asked to participate in a pilot randomized 
controlled trial. A separate informed consent will be used for the intervention study. 
 10.2   Subject Recruitment 
Healthy controls will be identified via Primary Care and adverts. Alternate Day Fasting subjects will 
be recruited by adverts and leaflets at Alternate Day Faster Events. 
10.3       Inclusion Criteria  
 Age between 35 and 65 years, 
 Body mass index in the range of 22.0 – 30.0 kg/m2,  
 Fasting blood glucose <110mg/dL (without medication) 
 LDL-cholesterol <180 mg/dL (without medication) 
 Blood pressure <140/90 mmHg (without medication) 
 Stable weight (change <± 10%) for 3 months immediately prior to the study, 
 No history of metabolic disorders or cardiovascular disease 
 No acute or chronic inflammatory disorder 
 No current medications to regulate blood sugar, blood pressure or lipids or hormones 
 No heavy drinking (more than 15 drinks/week) 
 No use of tobacco or recreational drugs within past 5 years 
 No dietary restrictions (e.g. vegetarianism and vegan) 
10.4     Exclusion Criteria  
 Known Malignancy 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 12 of 32 
Division of Endocrinology and Diabetology
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
 Women who are pregnant, breast-feeding or trying to become pregnant 
 History of any chronic disease process that could interfere with interpretation of study results 
 Women or men on hormonal supplementation or anti-conceptive hormonal medication for at 
least 2 months 
 Therapy with antidepressants within past 6 months 
 Regular therapy with acetylsalicylic acid 
 
10.5     Withdrawal / Drop out of subjects 
Each subject has the right to withdraw from the study at any time without prejudice or 
compromise to future care.  
The investigator may discontinue or withdraw the subjects under the following circumstances: 
- Significant protocol deviation 
- Significant non-compliance with treatment regime or study procedures 
- An adverse event that requires discontinuation of the study medication or results in inability 
to continue to comply with study procedures. If a subject is withdrawn due to an adverse 
event, the investigator will arrange for follow-up visits or telephone calls until the adverse 
event has resolved or stabilised. 
- Consent withdrawn 
- Lost to follow up 
- Any other situation that may, in the opinion of the investigator, make it unsafe or 
inappropriate for the subject to continue in the trial 
10.6      Payment to subjects 
   Reimbursement of reasonable travel expenses will be considered.   
 
11.    STUDY PROCEDURES  
11.1  Schedule of study procedures of the cohort study 
 
Visit 1 
Visit 2  
(9 ± 4 days after visit 1) 
Informed Consent 
X 
 
Inclusion/exclusion criteria 
X 
 
Demography, medical history 
X 
 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 13 of 32 
Division of Endocrinology and Diabetology
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Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
Concomitant medication 
X 
X 
Vital signs 
X 
X 
Physical examination 
X 
 
Blood sampling (FMD) 
X 
X 
Dual-energy X-ray absorptiometry (DXA) 
 
X 
Oral glucose tolerance test (OGTT) 
X 
X (ADF group only) 
Electrocardiography (ECG) 
X 
 
Questionnaires (FFQ, Physical Activity) 
 
X 
Retinal vessels analyser 
 
X 
Continuous Glucose monitoring (CGM) 
X 
 
Hand-Grip Test 
 
X 
Accelerometer (Movisens®) 
X 
 
Endothelial function (EndoPat 2000) 
 
X 
Intima media thickness (IMT) 
 
X 
Pulse wave analysis 
 
X 
Buccal mucosa sampling 
X 
 
24h blood pressure measurement 
X 
 
Sputum sampling 
X 
 
Faeces sampling 
X 
 
Echocardiography 
X 
 
Resting energy expenditure (REE) 
 
X 
Bioelectrical impedance analysis (BIA) 
 
X 
Dry eye assessment (optional) 
 
X 
Flow mediated dilatation (FMD) 
 
X 
11.2   Visit Schedule for the cohort study 
Visit 1 
- 
Sufficient time will be provided and subjects will be encouraged to discuss all queries with the 
research team 
- 
Informed consent for the study will be obtained by an investigator 
- 
Biobank informed consent will be obtained by an investigator 
- 
Check inclusion and exclusion criteria 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 14 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
- 
Demographics will be obtained: date of birth, gender, race, smoking status, alcohol consumption 
- 
Weight 
- 
Height 
- 
Waist-Hip Ratio 
- 
Medical history and standardized questionnaire on fertility outcomes 
- 
Concomitant medication  
- 
Vital signs: resting pulse and blood pressure  
- 
Blood sampling 
- 
ECG 
- 
Food Frequency Questionnaire and Ferriman-Galway Score/Hamilton-Norwood Scheme 
- 
Baseline blood tests (fasting): Full blood cell count, renal function tests, liver function tests, 
- 
Pregnancy test in female woman of childbearing age 
- 
Oral glucose tolerance test (OGTT)  
- 
Application of the 24h blood pressure device 
- 
Application of continuous glucose monitoring  
- 
Application of accelerometer (Movisens®) 
Visit 2 (9 ± 4 days after visit 1) 
- 
Vital signs, Weight 
- 
Physical examination 
- 
Concomitant medication review 
- 
Resting energy expenditure (REE) 
- 
Measurement of endothelial function (EndoPAT 2000) 
- 
Flow mediated dilatation (FMD) 
- 
Blood sampling  
- 
Oral glucose tolerance test (OGTT)  
- 
IMT measurement  
- 
Retinal vessel analyser 
- 
Hand Grip Test 
- 
Pulse wave analysis  
- 
Echocardiography 
- 
Collection and analysis of 24h blood pressure monitor 
- 
Collection and analysis of accelerometer 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 15 of 32 
Division of Endocrinology and Diabetology
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
- 
Removal of continuous glucose monitoring 
- 
Dual-energy X-ray absorptiometry (DXA) and hand grip test  
- 
Collection of sputum and faeces samples 
- 
Buccal mucosa sampling 
- 
Informed consent for RCT (healthy control group willing to participate in the RCT) 
- 
Bioelectrical impedance analysis (BIA) 
- 
Dry eye assessment (optional) 
 
Visit 3 (2 weeks before visit 4) → only those subjects participating in the pilot RCT 
- 
Application of continuous glucose monitoring  
 
Visit 4 (4 weeks ± 1 week after visit 2) → only those subjects participating in the pilot RCT 
- 
Vital signs, Weight 
- 
Physical examination 
- 
Concomitant medication review 
- 
Resting energy expenditure (REE) 
- 
Measurement of endothelial function (EndoPAT 2000) 
- 
Flow mediated dilatation (FMD) 
- 
Blood sampling  
- 
Food Frequency Questionnaire 
- 
Oral glucose tolerance test (OGTT)  
- 
Removal of continuous glucose monitoring 
- 
Collection of sputum and faeces samples 
- 
Buccal mucosa sampling 
- 
Bioelectrical impedance analysis (BIA) 
 
Visit 5 (2years ± 4 weeks after Visit 4) → → only those subjects participating in the pilot RCT 
 
- 
Vital signs, Weight 
- 
Physical examination 
- 
Concomitant medication review 
- 
Blood sampling  

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 16 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
- 
Food Frequency Questionnaire 
 
11.3  Schedule of study procedures of the pilot intervention trial 
 
Visit 3 
(14 ± 2 days after 
visit 2) 
Visit 4 
(4 weeks ± 1 week after 
visit 2) 
Visit 5 
(2years ± 4 weeks 
after Visit 4) 
Application of continuous glucose 
monitoring 
X 
 
 
Concomitant medication 
 
X 
X 
Vital signs 
 
X 
X 
Physical examination 
 
X 
X 
Blood sampling 
 
X 
X 
Oral glucose tolerance test (OGTT) 
 
X 
 
Continuous Glucose monitoring 
(CGM) 
 
X 
 
Endothelial function (EndoPat 2000) 
 
X 
 
Buccal mucosa sampling 
 
X 
 
Saliva sampling 
 
X 
 
Faeces sampling 
 
X 
 
Resting energy expenditure (REE) 
 
X 
 
Questionnaires  (FFQ, Physical 
Activity) 
 
X 
X 
Bioelectrical impedance analysis (BIA) 
 
X 
 
Flow mediated dilatation (FMD) 
 
X 
 
 
 
11.4   Laboratory results 
All laboratory results will be reviewed and the reports signed by the study physician who will 
record whether it is normal, abnormal but not clinically significant, or abnormal and clinically 
significant. In the latter case, the eligibility of the subject will be reviewed. 
 
 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 17 of 32 
Division of Endocrinology and Diabetology
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
12.   METHODS 
12.1   Oral glucose tolerance test (OGTT)  
Various insulin sensitivity indices derived from a frequently sampled, extended oral glucose tolerance 
test (OGTT) will be calculated accounting for the various weighting of hepatic and peripheral insulin 
sensitivity expressed by the different indices. Insulin, C-peptide and glucose will be measured before and 
15, 30, 60 and 120 minutes after ingestion of 75g glucose (®Glucoral 75 citron, Germania Pharmazeutika, 
Vienna). In addition, lipid-parameters (total cholesterol, triglycerides, HDL, LDL, free fatty acids) and a 
routine safety lab will be measured. The OGTT will be performed after an overnight fast (apart from 
water). A standard gauge cannula will be placed into a subcutaneous vein for blood sampling. In order 
to prevent blood clotting in the cannula and to keep the cannula working it will be occasionally flushed 
with sterile normal saline. A pre-meal blood sample will be taken (-5 mins) and then all subjects will be 
asked to drink the sugar solution over a period of 2-4 mins (time 0 min).  During the OGTT further blood 
samples will be taken at 15, 30, 60, and 120 minutes. The blood at each time point will be placed into a 
fluoride oxalate tube (1ml) for plasma glucose and into a serum tube for analyses of insulin and c-
peptide. 
The insulin sensitivity index will be calculated from the OGTT according to four different equations: the 
Matsuda-Index (ISOGTT): 𝐼𝑆𝑜𝐺𝑇𝑇=
10000
√(𝑔𝑙𝑢𝑐𝑜𝑠𝑒0∗𝑖𝑛𝑠𝑢𝑙𝑖𝑛0)∗(𝑚𝑒𝑎𝑛 𝑔𝑙𝑢𝑐𝑜𝑠𝑒∗𝑚𝑒𝑎𝑛 𝑖𝑛𝑠𝑢𝑙𝑖𝑛), 14 the  HOMA-IR 
(Homeostasis Model Assessment for Insulin Resistance): 𝐻𝑂𝑀𝐴−𝐼𝑅=
𝐹𝑃𝐺(𝑚𝑚𝑜𝑙𝑙
⁄ )∗𝐹𝑆𝐼(𝑈𝑙
⁄ )
22.5
     15 and the  
𝑄𝑈𝐼𝐶𝐾𝐼=
1
log(𝑖𝑛𝑠𝑢𝑙𝑖𝑛0)+log (𝑔𝑙𝑢𝑐𝑜𝑠𝑒0)  16. The last equation was proposed by Stumvoll et al. (insulin 
sensitivity index ISI): 𝐼𝑆𝐼= 0.222 −0.00333 ∗𝐵𝑀𝐼−0.0000779 ∗𝐼𝑛𝑠120 −0.000422 ∗𝑎𝑔𝑒 17. 
Beta-cell function will be estimated in the fasting state with 𝐻𝑂𝑀𝐴−𝛽=
20∗𝐼𝑛𝑠𝑢𝑙𝑖𝑛0
𝐺𝑙𝑢𝑐𝑜𝑠𝑒0−3.5    15 and during 
the OGTT with the Stumvoll-Index:  1𝑠𝑡 𝑝ℎ𝑎𝑠𝑒= 1283 + 1.829 ∗𝐼𝑛𝑠30 −138.7 ∗𝐺𝑙𝑐30 + 3.772 ∗
𝐼𝑛𝑠0     𝑝𝑙𝑢𝑠     2𝑛𝑑𝑝ℎ𝑎𝑠𝑒= 286 + 0.416 ∗𝐼𝑛𝑠30 −25.94 ∗𝐺𝑙𝑢𝑐30 + 0.926 ∗𝐼𝑛𝑠0 17  and the ratio 
of the incremental insulin to glucose response over the first 30min during the OGTT  
∆ 𝐼𝑛𝑠𝑢𝑙𝑖𝑛 (30)
∆ 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 (30) . 
12.2   Endothelial function  
Endo-PAT 2000 (Itamar Medical Ltd., Caesarea, Israel) will be used to measure endothelium-dependent 
vaso-reactivity as previously described 18. In brief, before measurements, the subjects will be in supine 
position for a minimum of 10 minutes, in a quiet, temperature-controlled room with dimmed lights. 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 18 of 32 
Division of Endocrinology and Diabetology
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Tel: ++43 (0) 316 380 1507 
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Probes will be placed on both index fingers and pulse wave amplitudes will be detected and recorded 
during the study. After a five-minute baseline measurement, arterial flow will be occluded using a cuff 
on the non-dominant arm. The cuff will be inflated to 60 mmHg above systolic pressure. After five 
minutes of occlusion, the cuff will be rapidly deflated to allow reactive hyperemia. Pulse wave amplitudes 
will be recorded again for at least five minutes. The ratio of the peripheral arterial tone (PAT) signal after 
cuff release compared with baseline will be calculated through a computer algorithm automatically 
normalizing for baseline signal and indexed to the contra lateral arm. The calculated ratio represents the 
reactive hyperemia index (RHI) 19.  
12.3   Bone densitometry and body composition  
Bone density scanning, also called dual-energy x-ray absorptiometry (DXA) or bone densitometry, is an 
enhanced form of x-ray technology that is used to measure bone loss. DXA measurement will be 
performed with a GE Lunar iDEXA (GE Healthcare, Waukesha, WI, US) full size for the purpose of 
estimating percentage body fat according to the departmental Standard Operating Procedure. Body 
regions are defined using standard anatomical partitions. Scan areas are analysed to determine lean 
mass, fat mass, bone mineral content, and total body composition. 
12.4   Laboratory measurements (Clinical and hormonal) 
Insulin and c-peptide will be measured by chemiluminescence on an ADVIA Centaur system (Siemens 
Healthcare Diagnostics, Eschborn, Germany). Fertility hormones (AMH (Anti-muellerian hormone), 
testosterone, cortisol, thyreotropin, triiodothyronine and thyroxine, estrogen, SHBG (sexual hormone 
binding globulin), LH (luteotropic hormone), FSH (follicle stimulating hormone) and 25(OH)vitamin D will 
be measured using automated analyzers: AMH by Beckmann-Coulter, Krefeld, Germany; testosterone, 
cortisol, thyreotropin,   triiodothyronine and thyroxine  by Siemens ADVIA Centaur, Eschborn, Germany;  
SHBG by Roche Diagnotics, Mannheim, Germany; estrogen, LH and FSH by Triturus, Biomedical 
Diagnostics, Antwerp, Belgium, 25(OH)vitamin D by iSYS, IDS, Boldon, U.K.) respectively. Samples for 
appetite hormones (Leptin, Ghrelin, PYY, and others) will be stored at -80°C until analysis after 
centrifugation. In addition, lactose intolerance (as defined by the locus LCT-13910) and vitamin D 
genotypes will be determined by standardized PCR from EDTA blood samples. FFA will be measured 
enzymatically (Wako Chemical, Neuss, Germany) on an Olympus AU640 (Olympus Diagnostica, Hamburg, 
Germany). Routine parameters will be determined using a cobas analyzer (Roche Diagnostics, 
Mannheim, Germany). 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 19 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
12.5   Laboratory measurements (Specialized Research – Aging Parameters) 
Molecular parameters of aging will be measured by a variety of already established methods at the Prof. 
Madeo’s laboratory at the Institute of Molecular Biosciences, University of Graz. Levels of different cell 
signaling and cell cycle proteins (p16, p21, p53, NCX, mTOR, Ras, Rb) will be measured using capillary-
based western blot system – Wes™ (Proteinsimple, San Jose, California, USA). Inflammatory markers, 
insulin signaling, as well as precise characteristics of molecular pathways will be analyzed using certified 
and commercially available V-PLEX Assays (Meso Scale Discovery, Rockville, Maryland, USA). Epigenetic 
changes (methylation and acetylation) will be analyzed via traditional western blotting. All methods 
based on antigen-antibody interaction will be done using commercially available antibodies, in order to 
assure better reproduction and optimal processes and procedures. Different “omics” (metabolomics, 
proteomics, acetylproteomics) as well as levels of different polyamines will be measured using HPLC-
coupled mass spectrometry (MS) or HPLC-coupled MS/MS methods.  Different parameters of cell death 
(apoptosis, stress resistance, mitochondrial damage) and damage caused by aging (telomere length, 
double strand breaks, chromosomal aberrations, heterochromatin changes) will be analyzed using flow 
cytometry systems BD Bioscience LSRFortessa and FACSAria IIu at the NAWI Graz Central Lab “Graz Cell 
Informatics and Analyses”. The telomerase activity will be measured by Human Telomerase Reverse 
Transcriptase (hTERT) ELISA Quantitation Kit (GenWay, San Diego, California, USA).  
12.6   Physical activity measurement 
The MoviSens-device consists of a 5.3×3×2-cm-sized body and can be fixed with a clip to the hip. During 
the measurements, the sensor will be placed on the right side of the subjects’ hip. The sensor is a three-
axial acceleration sensor with a range of ±8 g, a resolution of 12 bit and a sampling rate of 128 Hz. The 
recorded data from the sensor, including raw data from the acceleration sensor, can be displayed on a 
computer when connected to it via USB cable. Energy expenditure is displayed in steps of 1 s. Short time 
intervals allow monitoring spontaneous activities. The recognition of different activities is based on the 
extraction of mathematical and statistical features of the raw acceleration signal. The features are 
calculated for each segment of 4 s. Calculated features are, amongst others, maximum frequency, step 
count, and the number of mean crossings. These features are the input information of a decision tree 
which classifies the activity of the person. Activities that can be detected are rest (combination of lying, 
sitting, and standing); bicycle or ergometer; going upstairs; walking (combination of jogging, going 
downstairs, walking slow, normal, and fast); and unknown activity. According to information from the 
manufacturer, the decision tree was generated using data of approximately 300 subjects who performed 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 20 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
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University of Graz 
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Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
daily life activities. The accuracy of the activity recognition algorithm is discussed by Jatobá et al. 20. 
According to the detected activity, one of five different models is selected. 
12.7   Intima media thickness (IMT) 
The measurement of the common carotid artery intima and media combined layers as determined by 
high-resolution B-mode ultrasonography is widely recognized as a useful tool for early identification for 
systemic atherosclerosis. Intima media thickness (IMT) and plaque will be measured using high 
resolution ultrasound. IMT will be measured in the common carotid artery in a segment of 2 cm below 
the bulb. Near and far wall measurements of both sides will be combined to calculate mean IMT 21. 
Plaque burden will be quantified using the B-score according to the ACAPS protocol 22. 
12.8   Food frequency questionnaire (FFQ) and hormonal scores 
A self-administered, semi-quantitative FFQ was developed to assess usual food consumption within the 
German Health Examination Survey for Adults 2008-2011 (DEGS) 23. The relative validity of this 
questionnaire was studied among participants of another nationwide survey, the German National 
Nutrition Monitoring (NEMONIT) 24. The FFQ includes questions about the frequency and the amount of 
53 food items, consumed during the past four weeks. Frequency of consumption of food items was asked 
according to specified categories. In addition, the respondents had to indicate the portion size of the 
food items consumed in predefined answering categories. Pictures were used to aid the estimation of 
portion size for 33 food items. In total, 29 food groups will be presented by the food frequency 
questionnaire. Standardized fertility questionnaires are published medical history questions asking for 
menarche/menopause, menses regularity and childbirth/child wish in female and complementary data 
in male probands. Ferriman-Galway Score and Hamilton-Norwood Scheme are standardized 
questionnaires/schemes for female hirsutism or male baldness, respectively. Probands and/or medical 
doctors use pictograms for a scoring of female/male body hair. 
12.9   Non-invasive 24h ambulatory blood pressure monitoring 
Subjects wear the blood pressure device for a single 24-hours period. During this time, the device is 
programmed to inflate and record blood pressure at pre-specified intervals (usually every 15min during 
daytime hours and every 30 minutes during night-time hours), which provides approximately 50-75 
blood pressure recordings during the 24-hours period. Study participants are asked to keep an activity 
log throughout the 24-hour period so activities can mapped onto the blood pressure recordings. The 
process for educating subjects about the blood pressure device and setting up the test takes 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 21 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
approximately 10 minutes. Patients should undergo testing on a regular workday. They should be 
educated about the process of monitoring and the importance of maintaining an activity log. 
12.10    Resting energy expenditure (REE) 
The resting energy expenditure (REE) is a component of energy expenditure that is measured by indirect 
calorimetry (IC). Subjects must at least 30-minute rest after at least 8 hours of sleep and after at least 3 
hour fasting. During measurement, subject must be kept fully awake, lied down quietly, completely 
relaxed and breathing normally. The measurements took place for at least 30 minutes and were 
performed in standard neutral hospital room temperature. A breath mask will be placed over the head 
of the subject. Oxygen consumption and carbon dioxide production will be measured and energy 
expenditure will be calculated by the Weir formula 25. 
12.11   Hand Grip Test  
One of the most common methods of measuring muscle strength is the isometric grip strength test. We 
will measure isometric grip strength using a handgrip dynamometer (JAMAR®, Nottinghamshire, UK).  
The test will be performed in the sitting position with the shoulder of tested arm adducted, the elbow 
flexed at 90°, whereas the forearm and wrist were set in neutral position 26. The testing protocol consists 
of three maximal isometric contractions for 5 s, on both hands, with a rest period of at least 60 s and the 
highest value will be used for determination of maximal grip strength. The subjects will be instructed to 
squeeze the dynamometer as hard as possible. 
12.12   Pulse wave analysis 
Radial Pulse wave analysis (PWA) and carotid-femoral pulse wave velocity (PWV) will be performed using 
the Sphygmocor tonometry system (AtCor Medical, Sydney, Australia) and assessed as previously de-
scribed27. Participants will be in the supine position. Briefly, a central aortic pressure waveform will be 
derived from a measured radial artery pressure waveform. Aortic augmentation index (AIx) will be calcu-
lated as the difference between the first and second systolic peaks of the ascending aortic waveform 
divided by the difference between central aortic systolic blood pressure (SBP) and central aortic diastolic 
blood pressure (DBP). AIx will be standardized to a heart rate of 75 beats/minute (AIx75). To determine 
PWV, ECG-gated pulse pressure waveforms will be obtained sequentially over the right carotid artery 
and the right femoral artery. PWV will be calculated as the transit distance between the measurement 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 22 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
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Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
sites divided by the transit time computed as the time interval between the feet of the two pressure 
waves. 
12.13   Echocardiogram (ECG) 
A 12-lead ECG will be performed by a co-investigator or delegated staff. The ECG print out must be 
interpreted, signed and dated by a co-investigator. 
12.14   Buccal mucosa sampling 
Perform mouth wash with distilled water (three replicates) to remove traces of saliva and surface 
mucous. Then perform exfoliation within the two cheeks to maximize the cell sample and remove any 
trace unknown, which may be caused by sampling of only one cheek. The exfoliation should use a 
disposable brush similar to that used for Pap smear, performing 10 rotations of the brush against the 
inner wall of the cheeks starting in the centre and gradually increasing the circumference, producing a 
spiral effect to increase the sampling of a larger area and prevent erosion continuous of a single region. 
After exfoliation, the brushhead must be placed in a container (test tube) with 04ml of buffer containing: 
0.01 M Tris hydrochloride (Tris HCl), 0.1 M acid ethylenediaminetetraacetic (EDTA) and 0.02M Sodium 
Chloride (NaCl) with pH 6.8, turning in the way that the cells are released and displaced on the inner 
edge of the container 28. 
The micronucleus (MN) assay in exfoliated buccal cells is a useful and minimally invasive method for 
monitoring genetic damage in humans 29. The Buccal Micronucleus Cytome (BMCyt) assay is a minimally 
invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative 
potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological 
studies for investigating the impact of nutrition, lifestyle factors, genotoxin exposure and genotype on 
DNA damage, chromosome malsegregation and cell death. The biomarkers measured in this assay have 
been associated with increased risk of accelerated ageing, cancer and neurodegenerative diseases 30. 
12.15   Blood sampling and storage  
Over the study in those participating in the cohort study only in total approximately 150 ml of blood will 
be taken to determine the parameters outlined above, in those also participating in the RCT in total 
(cohort study and RCT) approximately 200 ml of blood will be drawn. A potential remainder of serum 
and plasma will be stored anonymised (patient number and visit number only) at -80°C in the Biobank 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 23 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
of the Medical University of Graz for potential future analyses. Therefore subjects will be asked to sign 
the informed consent of Biobank. 
12.16   Faeces sampling 
Sampling will be performed using two stool collection tubes by Sarstedt, Nümbrecht, Germany, using a 
stool collector (Süsse Stuhlfänger, Gudensberg, Germany). One tube will be used for native stool, the 
other prefilled with stool DNA-stabilizer for collection of DNA-stabilized stool specimen. Directions for 
safe and hygienic fecal collection will be delivered to the probands in words as well as in pictograms.  
Bacterial DNA will be extracted from saliva and stool samples using the MagNA Pure LC DNA Isolation Kit 
III (Roche). The 16S rRNA gene will be amplified in a PCR reaction and sequenced with next-generation 
sequencing technology (Roche Genome Sequencer FLX or Illumina MiSeq) and interpreted by the 
respective software analyzers. 
12.17   Saliva sampling 
Saliva will be collected by Salitubes (DRG Instruments Marburg, Germany) using a routine method for 
saliva collection and dedicated tubes as well as standardized instructions for the collection.  Hormones 
and salivary microbiome will be analysed by methods described in 12.4 (adapted ELISAs by BSM, Vienna, 
Austria) and 12.16, respectively.  
12.18  Continuous glucose monitoring 
Minimally invasive CGMS will be performed using the Abbott FreeStyle Flash or Medtronic Ipro 2 glucose 
monitoring device. This system eliminates the need for routine finger pricks, reading glucose levels 
through a sensor that can be worn on the back of the upper arm for up to 7-14 days. In addition, no 
finger prick calibration is needed—a key differentiator from current continuous glucose monitoring 
systems. Both, Abbott´s FreeStyle Flash and Medtronic Ipro 2 glucose monitoring systems, consist of a 
small, round sensor—approximately the size of a two Euro coin—worn on the back of the upper arm, 
which measures glucose every minute in interstitial fluid through a small (5mm long, 0.4mm wide) 
filament that is inserted just under the skin and held in place with a small adhesive pad. A reader is 
scanned over the sensor to get a glucose result painlessly in less than one second. Scanning can take 
place while the sensor is under clothing, making testing more discreet and convenient. Each scan displays 
a real-time glucose result, a historical trend and the direction the glucose is heading. The reader holds 
up to 90 days of data, providing a historical snapshot of glucose levels over time. The Medtronic devices 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 24 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
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Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
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requires regular (at least twice daily) calibration measurements with a common glucose measurement 
device. 
In the cohort study the CGM sensor will be used once (14 days, at visit 1) and in those subjects 
participating in the pilot intervention trial, CGM will be used a second time (another 14 days, CGM will 
be started at visit 3). 
12.19  Physical examination 
Physical examination will be performed at visit 1 and include examination of: 
- 
General appearance 
- 
Head, ears, eyes, nose, throat, neck 
- 
Cardiovascular system  
- 
Respiratory system 
- 
Skin 
- 
Thyroid gland 
- 
Musculoskeletal system 
- 
Central and peripheral nervous system  
12.20  Vital signs 
Systolic and diastolic blood pressure measured in mmHg will be collected at both visits to the trial site. 
The blood pressure should be measured in a sitting position, with legs uncrossed, the back and arms 
supported. Subjects should be sitting for at least five minutes before the measurement is taken and 
subjects should not talk during the measurement. Pulse as beats per minute will be recorded after resting 
for five minutes in a sitting position.  The pulse should be measured at both study visits to the trial site. 
12.21  Echocardiography 
Echocardiographic examination will be performed with a Vivid 7 (GE Healthcare, Chalfont St Giles, UK). 
For acquisition of images in the parasternal view the participant will be placed in the steep left-lateral 
decubitus position. The participant’s left arm will be raised. Images will be acquired during quiet 
respiration. Image rate will be at least 30 frames per second. 
12.22   International physical activity questionnaire (IPAQ) 
IPAQ will be used in the current study. This tool was developed for measuring physical activity in 1998 
and was followed by extensive reliability and validity testing undertaken across 12 countries (14 sites) 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 25 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
during 200031, 32. The final results suggested that this tool had acceptable measurement properties for 
use in many settings and in different languages and was suitable for national population-based 
prevalence studies of participation in physical activity. The short version of IPAQ tool will be used in the 
current study. The tool includes a set of 4 questionnaires. The questions asked about the time spent 
being physically active in the last 7 days.  
12.23   Bioelectrical impedance analysis (BIA) 
Bioelectrical impedance analysis is a fast, reliable, affordable and non-evasive technique to measure 
body composition. Its accuracy and dependability have been scientifically proven 33, 34 and clinically 
demonstrated time and time again. It uses the fact that tissue types differ in electric conductivity. Bones 
and fat, for instance are bad conductors. Skeletal muscles on the other hand, due to their water and 
electrolyte content, have much lower impedance. BIA makes it possible to precisely determine the 
distribution of the individual components, e.g. fluids (“water”), muscle mass and fatty substance in the 
body. 
12.24 Dry eye assessment  
Recent findings show that dry eyes syndrome (“Sicca” syndrome) is tightly regulated by the metabolic 
and inflammatory factors both on the eye surface and on a systemic level during aging 35. Since we 
propose intermittent fasting besides caloric restriction 36 as one of the most significant interventions into 
the human metabolism, we will conduct the preliminary evaluation of the effects of intermittent fasting 
on several main clinical parameters of the Sicca syndrome. As part of our trial we will investigate:  
- 
redness of cornea and the stability of basal tear film by using Oculus Keratograph 5M 
- 
the degree of epithelial damage on the eye surface by fluorescein staining 
- 
the tear volume by Schirmer’s test I 
- 
the quantity and the quality of the meibomian gland’s secretion 
- 
the metabolome of basal tear fluid by mass spectrometry 
- 
the risk factors and symptoms of the Sicca syndrome by using the OSDI (Occular Surface Diseases 
Index) questionnaire. 
All procedures are carried out under non-invasive conditions and lead to no influence of the eye function 
of the participant, both in short and long term. By analysing the effects of intermittent fasting on the 
development of dry eyes syndrome, we want to examine the potential of such a treatment as a 
preventive and possibly even interventional method to sustain and improve eye health during aging.  

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 26 of 32 
Division of Endocrinology and Diabetology
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12.25   Flow mediated dilatation (FMD) 
Endothelium-dependent FMD following reactive hyperaemia is examined in the brachial artery according 
to the guidelines described by Corretti et al 37. Using high resolution ultrasound measurements of the 
right brachial artery are taken after resting and after cuff deflation (250 mmHg for 5 minutes) of the 
forearm. Scans of the brachial artery are taken proximal to the antecubital fossa with simultaneous ECG 
recording. Diameter measurements are taken from one media-adventitia interface to the other for three 
to four times at baseline (the average is taken for calculating the baseline diameter) and three times 
after 45 to 60 seconds after cuff deflation following reactive hyperaemia. FMD-diameter is calculated as 
the average of the three diameter measurements following reactive hyperaemia. The dilatation is 
calculated as the percent change in diameter compared to baseline.  
 
12.26   Data analysis and sample size estimation 
Data analysis 
Statistical Analyses for primary and secondary outcomes as well as for baseline values are done in a 
descriptive and exploratory way. 
Continuous variables will be summarized with standard descriptive statistics tables and represented 
graphically with displays such as box plots and histograms, and categorical variables will be described by 
frequency tables and standard graphical displays like bar plots. Scatterplots are used to illustrate possible 
correlations between the variables.  
To examine differences between groups at baseline and following visits, Student’s T-tests or Mann-
Whitney U tests and chi-square tests will be used as appropriate. Comparisons within groups will be 
performed using paired T-test or Wilcoxon rank-sum test. A p-value of <0.05 is considered to indicate 
statistical significance, whereas appropriate correction for multiple testing like Bonferroni Correction or 
false discovery rate (Benjamini and Hochberg) will be applied. To investigate multiple relations 
generalized linear mixed models will be built to investigate changes between groups and over time, as 
the physiological changes are represented in various parameters. For high-dimensional “omics-” data 
appropriate multivariate modelling like Random Forests and Partial Least Squares Regression will be 
used to elucidate differences between groups and time-points concerning potential molecular markers 
regarding aging effects. 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 27 of 32 
Division of Endocrinology and Diabetology
Medical University of Graz
Auenbruggerplatz 15
Tel: ++43 (0) 316 385 13270
Fax: ++43 (0) 316 385 14332
Institute of Molecular Biosciences 
University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
A statistical analysis plan will be prepared and finalized before database lock. All statistical analysis will 
be done in R (version 3.0.1.). 
 
 
Sample size estimation 
Data on the effects of ADF on metabolic parameters, human physiology, aging process and molecular 
cellular processes in healthy subjects is very limited and a therefore an accurate sample size estimation 
impossible. However, we reviewed available data and used data on insulin sensitivity from a similar, 
albeit not entirely comparable study population with a HOMA-IR of 3 ± 1.1 (mean ± SD) 7. Assuming a 
power of 80% and a type I error rate of 5%, we would need 50 subjects per group to demonstrate an 
effect size of 15%, which has been demonstrated previously 38. Therefore the sample size of 50 subjects 
in the ADF and 50 subjects in the control group, as chosen for our cohort study, seems to be reasonable. 
In addition, previous publications have also suggested a sample size of 50 subjects per group for such 
types of studies with insufficient details about the outcome parameters required for a sample size 
estimation39.  Subjects in the control group will be asked to participate in a pilot, randomized controlled 
trial, where they are randomized to either ADF or continuing in the control group. We expect an 
acceptance rate of about 40%, hence a sample size of about 10 participants per group. 
 
12.27   Markers of cardiovascular risk 
Serum Aliquots of 40 subjects will be used for miRNA-analyses (atherosclerosis-related intracellular 
signals) regarding cardiovascular risk  between Japanese and Austrian cohorts (in cooperation with Ichiro 
Wakabayashi, MD PhD; Hyogo College of Medicine; Japan). 
 
 
13.    STUDY MONITORING 
A risk-based approach will be taken to determine the frequency of study monitoring for the study which 
will be agreed with the Principal Investigator. Monitoring will be undertaken according to ICH GCP and 
the study monitoring plan. The study monitor will be suitably trained, qualified and experienced to 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 28 of 32 
Division of Endocrinology and Diabetology
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University of Graz 
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perform this task. Data will be evaluated for compliance with the protocol and accuracy in relation to 
source documents.  
 
 
 
14.    ETHICS 
The Investigator will ensure that this study is conducted in accordance with the principles of the 
Declaration of Helsinki, GCP-ICH and according to the protocol and the requirements of the concerned 
regulatory authorities.  The measurements taken are either non-invasive or minimally invasive (blood 
sampling). Therefore we consider this trial as low risk for participants. 
14.1   Informed Consent 
- 1. Informed consent will be obtained for all participants in the study by the study physician. 
- 2. Written versions of the subject information and Informed consent will be presented to the 
subjects detailing:  
 
The exact nature of the study 
 
The implications and constraints of the protocol 
 
The known side effects and any risks involved in taking part.   
- 3. It will be clearly stated that the subject is free to discontinue their participation in the study 
at any time for any reason without prejudice to future care, and with no obligation to give 
the reason for withdrawal. 
- 4. The subject will be allowed as much time as wished to consider the information, and the 
opportunity to question the Investigator, their usual care provider or other independent 
parties to decide whether they will participate in the study.  Written Informed Consent will 
then be obtained by means of subject dated signature and dated signature of the person who 
presented and obtained the informed consent.  
- 5. A copy of the signed Informed Consent will be given to the subjects. The original signed 
form will be retained at the study site. 
-  
- Every subjects participating in the cohort study will need to sign the cohort study informed 
consent and in addition the biobank informed consent. Those subjects willing to participate 
in the pilot RCT will be asked to sign an RCT specific informed consent in addition. 
 

 
Protocol InterFAST  V 1.9                                                                                                                                                           Page 29 of 32 
Division of Endocrinology and Diabetology
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University of Graz 
Humboldstrasse 50/EG 
Tel: ++43 (0) 316 380 1507 
Fax: ++43 (0) 316 385 9016 
14.2    Ethical and Regulatory Approvals 
The protocol, informed consent form, participant information sheet and any proposed advertising 
material will be submitted to the Ethics Committee of the Medical University of Graz, Austria.   
The Investigator will submit and, where necessary, obtain approval from the above parties for all 
substantial amendments to the original approved documents.  
15.    FINANCE 
This study will be jointly funded by the Institute of Molecular Biosciences, University of Graz and  the 
Division of Endocrinology, Medical University of Graz, Austria. 
16.    INSURANCE 
Participant insurance according to legal requirements will be contracted. 

 
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Division of Endocrinology and Diabetology
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Medical University of Graz
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University of Graz 
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