Alternate Day Fasting Combined With Exercise for the Treatment of Non-alcoholic Fatty Liver Disease (NAFLD)
Phase: NA
Conditions: Non-Alcoholic Fatty Liver Disease, Obesity
Sponsor: University of Illinois at Chicago

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Alternate day fasting combined with exercise  
for the treatment of non-alcoholic fatty liver disease (NAFLD) 
  
 
 
 
Principal investigator: 
Krista A. Varady, Ph.D. 
Associate Professor 
Department of Kinesiology and Nutrition 
University of Illinois, Chicago 
1919 West Taylor Street, Room 532 
Chicago, Illinois, 60612 
Tel: 312-996-7897 
Email: varady@uic.edu 
 
Study location: 
Human Nutrition Research Unit 
1919 West Taylor Street, Room 121C 
Chicago, Illinois, 60612 
 
Sponsor: 
NIH (NIDDK) R01 DK119783 
 
Version 13 
 
June 15, 2023 
 
 
 
 

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TABLE OF CONTENTS  
 
 
Table of Contents 
 
 
 
 
 
2 
List of Abbreviations  
 
 
 
 
 
3 
 
1.0 
Project Summary/Abstract 
 
 
 
4 
2.0 
Background/Scientific Rationale 
 
 
5 
3.0  
Objective/Aims 
 
 
 
 
5 
4.0 
Eligibility 
 
 
 
 
 
6 
 
 
4.1 Inclusion criteria 
 
 
 
 
6 
4.2 Exclusion criteria 
 
 
 
 
6 
5.0 
Subject Enrollment 
 
 
 
 
6 
6.0  
Study Design and Procedures  
 
 
7 
7.0 
Expected Risks/Benefits 
 
 
 
11 
8.0 
Data Collection and Management Procedures  
12 
9.0 
Data Analysis  
 
 
 
 
12 
10.0 
Quality Control and Quality Assurance  
 
12 
11.0  
Data and Safety Monitoring 
 
 
 
12 
12.0  
Statistical Considerations 
 
 
 
13 
13.0  
Regulatory Requirements 
 
 
 
14 
 
13.1 Informed Consent  
 
 
 
14 
 
13.2 Subject Confidentiality 
 
 
 
14 
 
13.3 Unanticipated Problems 
 
 
 
14 
14.0  
References 
 
 
 
 
 
14 
 
 
 
 
 
 

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LIST OF ABBREVIATIONS 
 
 
 
 
ADF 
 
Alternate day fasting 
BIA 
 
Bioelectrical Impedance analysis 
CRP                      C-Reactive Protein 
DXA 
 
Dual-energy X-ray Absorptiometry 
FGF-21  
Fibroblast growth factor-21 
IL-6 
 
Interleukin-6 
MRS 
 
Magnetic resonance spectroscopy 
NAFLD  
Non-alcoholic fatty liver disease 
OGGT  
Oral glucose tolerance test 
PI 
 
Principal investigator 
RBP4 
 
Retinol binding protein 4 
SHBG  
Sex hormone binding globulin 
TNF-a  
Tumor necrosis factor-alpha  
 

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1.0 Project Summary/Abstract 
 
Background: Approximately 65% of obese individuals have non-alcoholic fatty liver disease (NAFLD), and this 
condition is strongly related to the development of insulin resistance and diabetes. Innovative lifestyle strategies 
to treat NAFLD are critically needed. Alternate day fasting (ADF) has been shown in animals to reduce hepatic 
steatosis and improve hepatic insulin sensitivity, but these findings have yet to be confirmed in human subjects. 
ADF consists of a “feast day” where individuals are permitted to consume food ad libitum, alternated with a 
“fast day” where individuals consume 25% of their usual intake (~500 kcal). We performed a pilot study to 
evaluate the effects of ADF combined with exercise, versus ADF or exercise alone, on hepatic parameters in 
obese patients. Our results show that the combination of ADF plus exercise produced greater reductions in 
alanine aminotransferase (ALT; an indirect marker of hepatic steatosis), compared to ADF alone, or exercise 
alone, after 12 weeks. Greater decreases in insulin resistance, HbA1c, LDL cholesterol, and more pronounced 
increases in HDL cholesterol, were observed in the combination group versus individual interventions. Data from 
our pilot trial also suggest that these decreases in insulin resistance may be mediated in part by changes in 
hepatocyte-derived hormones (hepatokines) that occur with liver fat reduction. Although these pilot findings are 
very promising, these data still require confirmation by a well powered longer-term (24 week) clinical trial. 
Hypotheses: The present proposal will test the following hypotheses: (1) The combination group (ADF plus 
exercise) will experience greater reductions in hepatic steatosis (measured by magnetic resonance spectroscopy; 
MRS) when compared to ADF or exercise alone; (2) The combination group will experience greater 
improvements in hepatokine profile (fetuin-A, fetuin B, FGF-21, RBP4, selenoprotein P, SHBG, adropin) when 
compared to ADF or exercise alone; (3) The combination group will experience greater improvements in hepatic 
insulin sensitivity, insulin resistance and HbA1c and other metabolic disease risk variables (fasting glucose, 
fasting insulin, triglycerides, LDL cholesterol, blood pressure, inflammatory parameters) when compared to ADF 
or exercise alone. Methods: To test these objectives, a 24-week randomized, controlled, parallel-arm feeding 
trial will be implemented. Obese individuals with NAFLD (n = 360) will be randomized to 1 of 4 groups: (1) ADF 
(fast day: 25% energy intake, feed day: ad libitum fed, no exercise), (2) exercise (ad libitum fed, training 5 
days/week), (3) combination (ADF plus exercise), and (4) control (ad libitum fed, no exercise). Significance: If the 
aims of this application are achieved, this study will be the first to show that the combination of alternate day 
fasting plus aerobic exercise is an effective non-pharmacological therapy to reduce hepatic steatosis, improve 
hepatic insulin sensitivity and prevent the progression to type 2 diabetes in NAFLD patients. 
 
 
 

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2.0 Background/ Scientific rationale 
 
Nonalcoholic fatty liver disease (NAFLD) is characterized by an accumulation of fat in the liver (not resulting from 
excessive alcohol consumption). Approximately 65% of obese individuals have NAFLD, and this condition is 
strongly related to the development of insulin resistance and type 2 diabetes.1-4  While certain pharmacological 
agents have been shown to reduce liver fat (i.e. thiazolidinediones), there is mounting concern regarding the 
safety and weight-gaining effects of these compounds.5,6 In light of this, recent research has focused on non-
pharmacological lifestyle therapies to reduce hepatic steatosis, such as daily calorie restriction combined with 
aerobic exercise. Evidence from clinical trials suggest that this combination is an effective lifestyle therapy 
improve liver fat content and hepatic insulin sensitivity.7,8 
 
More recently, it’s been shown that intermittent fasting may produce even greater improvements in hepatic 
steatosis and hepatic insulin sensitivity, when compared to conventional calorie restriction. For instance, 
intrahepatic lipid accumulation was lower and insulin sensitivity was higher in mice fasted every other day, when 
compared to mice who were energy restricted every day.9 Moreover, data from human trials show that obese 
adults experience greater decreases in insulin and insulin resistance with intermittent fasting versus daily 
restriction.10,11 These findings suggest that intermittent fasting may be a more effective diet therapy to reduce 
hepatic steatosis and improve insulin sensitivity, when compared to daily calorie restriction.  
 
We recently performed a pilot study to evaluate the effects of alternate day fasting (ADF) and exercise, alone or 
combined, on various hepatic parameters in obese adults. ADF consists of a “fast day”, in which 25% of energy 
needs (~500 kcal) are consumed, alternated with a “feed day”, in which food is consumed ad libitum. Results 
from our pilot study show that the combination of ADF plus exercise produced greater reductions (-21 ± 9%) in 
alanine aminotransferase (ALT; an indirect marker of hepatic steatosis), compared to ADF alone (-13 ± 4%), or 
exercise alone (-16 ± 7%) after 12 weeks. Greater decreases in insulin resistance, HbA1c, LDL cholesterol, and 
more pronounced increases in HDL cholesterol, were observed in the combination group versus individual 
interventions. Data from our pilot trial also suggest that these decreases in insulin resistance by ADF plus 
exercise may be mediated in part by changes in hepatocyte-derived hormones (fetuin-A and FGF-21) that occur 
with liver fat reduction.12 Although these pilot findings are very promising, these data still require confirmation 
by a larger-scale (n = 360), longer-term (24 week) clinical trial. Accordingly, the specific aims of this study are: 
 
3.0 Objective/Aims 
 
Specific aim 1: To compare the effects of a combination intervention (ADF and aerobic exercise) versus ADF or 
exercise alone on hepatic steatosis over 24-weeks in prediabetic obese NAFLD subjects. 
Hypothesis 1: The combination group will experience greater reductions in hepatic steatosis (measured 
by MRS) when compared to the ADF group and the aerobic exercise group. 
 
Specific aim 2: To compare the effects of a combination intervention (ADF and aerobic exercise) versus ADF 
alone or aerobic exercise alone on hepatokine profile over 24-weeks in prediabetic obese NAFLD subjects. 
Hypothesis 2: The combination group will experience greater decreases in hepatokines with negative 
metabolic actions (fetuin-A, fetuin B, FGF-21, RBP4, selenoprotein P) and greater increases in 
hepatokines with positive metabolic actions (SHBG, adropin) when compared to the ADF group and the 
exercise group. 
 
Specific aim 3: To compare the effects of a combination intervention (ADF and aerobic exercise) versus ADF 
alone or aerobic exercise alone on hepatic insulin sensitivity, insulin resistance, HbA1c, and other metabolic 
disease risk variables over 24-weeks in prediabetic obese NAFLD subjects. 
Hypothesis 3: The combination group will experience greater improvements in hepatic insulin 
sensitivity, insulin resistance and HbA1c and other metabolic disease variables (fasting glucose, insulin, 
plasma lipids, blood pressure, TNF-a, IL-6) when compared to the ADF group and the exercise group. 
 
 
 

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4.0 Eligibility  
 
Subject eligibility will be assessed and determined by Kelsey Gabel M.S., R.D., or key research personnel 
identified in Appendix P. Screening will be conducted over Zoom and at the Human Nutrition Research Unit 
(HNRU) located in the Applied Health Sciences Building (1919 West Taylor St, Room 121C).  
 
4.1 Inclusion criteria:  
• 
Age between 18 to 65 years old 
• 
BMI between 30.0 and 59.9 kg/m2 
• 
NAFLD (hepatic steatosis ≥ 5% previously diagnosed) and ALT > 15 U/L 
• 
Prediabetic (fasting glucose: 100-125 mg/dl, HbA1c: 5.7-6.4%) or normoglycemic (fasting glucose: <100 
mg/dl, HbA1c: <5.7%) 13 
• 
Sedentary (<20 min, 2x/week of light activity at 3-4 metabolic equivalents (METs) for 3 mo prior to study) 14 
 
4.2 Exclusion criteria: 
• 
Have chronic liver disease other than NAFLD (hepatitis B or C, primary biliary cirrhosis, sclerosing cholangitis, 
autoimmune hepatitis, hemochromatosis, Wilson’s disease, α1-antitrypsin deficiency) 
• 
Consume excessive amounts of alcohol (Michigan Alcohol Screening Test score > 4) 15 
• 
Have a history of known cardiovascular, pulmonary or renal disease 
• 
Diagnosed T1DM or T2DM  
• 
Are not weight stable for 3 months prior to the beginning of study (weight gain or loss > 4 kg) 
• 
Are claustrophobic or have implanted metallic/electrical devices (e.g. cardiac pacemaker, neuro-stimulator) 
• 
Are not able to keep a food diary or activity log for 7 consecutive days  
• 
Are taking drugs that induce steatosis (e.g. corticosteroids, estrogens, methotrexate, Ca channel blockers)  
• 
Are taking drugs that benefit NAFLD (e.g. betaine, pioglitazone, rosiglitazone, metformin, or gemifibrozil) 
• 
Are taking drugs that influence study outcomes (weight loss, lipid-lowering, glucose-lowering medications) 
• 
Are perimenopausal or have an irregular menstrual cycle (menses that does not appear every 27-32 days) 
• 
Are pregnant, or trying to become pregnant 
• 
Are smokers 
 
5.0  Subject enrollment 
 
Independently living subjects from the Chicago area will be recruited by flyers posted around the University of 
Illinois at Chicago (UIC) campus. ResearchMatch.org will also be used to recruit participants. Subjects will also be 
recruited from the Hepatology Clinic at the University of Illinois Medical Center by Dr. Sean Koppe MD. During 
clinical visits with these NAFLD patients, Dr. Koppe will describe the study to each of these individuals and 
distribute a flyer and contact information to those who are interested. Study procedures will be conducted at 
the subject’s home. 
 
COVID screening precautions: The study coordinator will call the subject prior to each on-campus appointment 
and screen the subject using the UI Health COVID screening form. If the subject passes this screening, they will 
be invited for an in-person visit. When they arrive on campus, the subject will be once again screened using the 
UI Health COVID screening form. Their temperature will also be checked using a no-touch digital thermometer. 
All research personnel and participants will be required to wear masks during each appointment. Only one 
research staff will be present to limit exposure. This screening procedure will be implemented every time the 
subject comes to campus for a study visit.  
 
Each subject will attend an online screening appointment and an in-person appointment. During the online 
appointment: Subjects will review the consent form with the Study Manager, Kelsey Gabel, MS, RD, over Zoom. 
The subject will then sign the consent form using Adobe Sign. After signing the consent form, the subject will be 
screened via the approved questionnaire, which will assess eligibility based on the requirements listed above. If 

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the subject is eligible for the study based on the questionnaire, they will be invited for an in-person 
appointment. During the in-person appointment: The subject will visit the HNRU to have these measurements 
taken: body weight and height (for BMI), a blood draw to assess HbA1c, ALT and fasting glucose, a pregnancy 
test, and the Michigan Alcohol Screening Test (for alcohol consumption). The Study Manager will also email a 7-
day food record to the subject, and provide detailed instructions on how to complete the record. The subject 
will be asked to return the food record within 10 days.  
 
Subjects who meet all the inclusion/exclusion criteria will be invited to participate in the study. Eligible subjects 
will be randomized by way of a stratified random sample. The sample frame will be divided into strata based on 
BMI, sex, and age. Subjects from each stratum will then be randomized to 1 of 4 groups: 1) ADF, 2) aerobic 
exercise, 3) combination (ADF plus aerobic exercise), or 4) control. A total of n = 360 subjects (n = 90 per 
intervention group) will be recruited. All the subjects will have the right to withdraw from the study at any 
time. 
  
6.0 Study design and procedures 
 
A 24-week randomized, controlled, parallel-arm feeding trial will be implemented to test the effects of ADF and 
exercise, alone and in combination, on hepatic steatosis/fibrosis, hepatokines, hepatic insulin sensitivity, and 
other metabolic disease variables. Obese individuals with NAFLD (n = 360) will be randomized to 1 of 4 groups: 
(1) ADF (fast day: 25% energy intake, feed day: ad libitum fed, no exercise), (2) exercise (ad libitum fed, training 
5 days/week), (3) combination (ADF plus exercise), and (4) control (ad libitum fed, no exercise). Changes in 
hepatic steatosis will be measured by MRS, and hepatic insulin sensitivity will be assessed by OGTT testing and 
the Matsuda Index. All study activities will be carried out at the Human Nutrition Research Unit (HNRU) located 
in the Applied Health Sciences Building (1919 West Taylor St, Room 121C). 
 
Diet and exercise interventions  
 
Group 
Diet protocol 
Exercise protocol 
 
Combination 
(n = 45) 
Fast day: 25% energy intake  
Feed day: Ad libitum fed  
 
5 days/week, supervised 
60 minutes, Intensity: 85% HRmax 
ADF 
(n = 45) 
Fast day: 25% energy intake  
Feed day: Ad libitum fed  
 
No exercise 
Exercise 
(n = 45) 
 
Every day: Ad libitum fed  
 
5 days/week, supervised  
60 minutes, Intensity: 85% HRmax 
 
Control 
(n = 45) 
Every day: Ad libitum fed  
 
No exercise 
 
Diet protocol  
 
ADF group and combination groups: These participants will consume 25% of their baseline energy needs on 
the "fast day” and eat ad libitum at home on alternating “feed days”.  
 
Fast day (week 1-4): All fast day meals will be provided to subjects during week 0-4. Meals will be prepared 
in the metabolic kitchen of the HNRU. The diets will be provided as a 3-d rotating menu comprised of typical 
American foods. Meals will be formulated on the basis of the American Heart Association (AHA) guidelines 
(30% kcal from fat, 15% kcal from protein, and 55% kcal from carbs). All meals will be consumed outside of 
the research center. Fast day (week 5-24): At week 5, fast day meals will no longer be provided to subjects. 
Instead, subjects will meet one-on-one with a dietician for 60 min to learn how to self-select healthy foods 

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to stay within the calorie limits on the fast day. Feed day: On the feed day, subjects will eat ad libitum at 
home. 
 
Exercise group and control groups: These participants will be asked to maintain their regular eating habits 
during the trial. They will eat ad libitum at home every day, and will not receive any packaged meals or 
dietary counseling. 
 
 
Exercise protocol  
 
Exercise group and combination groups: Only combination and exercise groups will partake in the exercise 
training. All training sessions will be conducted at the subject’s home. Subjects will participate in a 
supervised aerobic exercise program 5 times per week, 40-60 min/d, for 24 weeks.16 Exercise will be 
performed by watching YouTube aerobic exercise (Zumba) videos at home. The video links will be provided 
by the study coordinator prior the exercise session. The coordinator conducting the study session on Zoom 
will have immediate knowledge of the subject's location so that they can relay this information to 
emergency personnel if needed. During the week 1 sessions, subjects will exercise for 40 minutes at a 
moderate intensity (65% heart rate max (HRmax)). During the week 2 sessions, subjects will train for 50 
minutes at a higher intensity (75% HRmax). For weeks 3-24, subjects will train for 60 minutes at an even 
higher intensity (85% HRmax). The participants will wear heart rate monitors (Fitbit Alta HR, Boston, MA) 
during each training session to provide visual feedback of their individualized target heart rate. Fitbit 
monitors will be distributed to subjects so that they can monitor their heart rate at home. Compliance with 
the training: A research assistant will be present to supervise all the training sessions over Zoom. All 
sessions will be pre-scheduled with the research assistant so the participant knows when to enter the Zoom 
meeting to perform the activity. Exercise will be carefully documented through regular online attendance at 
the training sessions. Subjects will be permitted to choose the day and time slot that works best for them. If 
a subject misses their session, they will be required to make up for it that same week. Subjects will be 
permitted to miss 5 sessions total during the 24-week trial.  
 
ADF group and control groups: ADF and control subjects will be asked to maintain their regular daily activity 
habits during the study. They will not participate in the exercise protocol. 
 
Control group protocol: Controls will be instructed to maintain their weight throughout the trial, and not to 
change eating or physical activity habits. Controls will not receive packaged meals or dietary counseling. Controls 
will visit the research center at the same frequency as the treatment groups (for outcome measurements) to 
control for investigator-interaction bias between groups. Controls will be offered free weight loss diet 
counseling (4 sessions) at the conclusion of the study by Kelsey Gabel, MS, RD.  
 
Subject compensation. Each subject will receive $500 upon completion of the trial. Compensation will be 
prorated (please see Initial Review application for details). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Summary of intervention groups and key outcome measures 
 
 
 
 
               

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Study activities  
 
Study activities 
Study visits (week of study) 
 
Screen 
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Informed consent 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening 
questionnaire 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Michigan Alcohol 
Screening test 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test 
• 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Body weight (at 
home) 
• 
• 
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• 
• 
• 
• 
• 
• 
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• 
• 
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• 
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• 
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• 
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Blood draw 
• 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Blood pressure/ 
heart rate 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Body composition 
(BIA) 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Waist 
circumference 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Body composition 
(DXA) 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Activity monitor 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Hepatic insulin 
sensitivity (OGTT)  
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Hepatic steatosis 
(MRS) 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Food record 
• 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Sleep 
questionnaires 
 
• 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
 
 
 
 
 
• 
Time per visit: 
1 
4 
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0 
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4 

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Body weight, blood pressure, heart rate: Body weight measurements will be taken weekly with subjects 
wearing light clothing and without shoes using a balance beam scale at home. Digital scales will be provided 
to each subject. Height will be assessed using a wall-mounted stadiometer at week 1. BMI will be assessed 
as kg/m2. Blood pressure and heart rate will be assessed at week 1, 13, 24.  
 
Body composition: Dual energy X-ray absorptiometry (DXA) will be performed on all subjects at week 1, 13, 
and 24 (iDXA, GE Inc) to assess fat mass, fat free mass, and visceral fat mass. Percentage body fat will be 
assessed in triplicate using a tetra-polar bioelectrical impedance analyzer each week (Omron HBF-500; 
Omron Health Care, Bannockburn, IL). Waist circumference will be measured weekly. 
 
Hepatic steatosis by magnetic resonance spectroscopy (MRS): MR scans will be performed at the Center 
for Magnetic Resonance Research (CMRR). MR images and spectrum will be acquired using a 3.0 T MR 
scanner. MR examinations will be performed by experienced MR technologists. Subjects will arrive at the 
CMRR in the morning following an overnight (8 h) fast. Total hepatic triglyceride content will be assessed at 
week 1, 13 and 24 by ¹H MRS. A body array MRI coil will be affixed to each subject’s back and aligned with 
the subject’s spine and shoulders for accurate repositioning of repeat scans. Each subject will be positioned 
face down and head first in the scanner on a memory foam mattress to minimize respiratory motion. An 8 
cm³ voxel is positioned within the right posterior lobe of the subject’s liver with guidance from the high-
resolution localization images. Manual shimming will be performed to a line width of ~40Hz to ensure high 
quality spectra. MR spectra with and without water suppression will be acquired with a single-voxel PRESS 
acquisition with a long repetition time (TR=5000ms), and a short echo time (TE=30ms) to limit the effects of 
magnetic relaxation. 17,18 The acquisition will be obtained with 32 averages to obtain sufficient signal, and 
the data will be Fourier-Transformed, filtered, baseline corrected, and phased. Areas of resonances from 
protons of water and methylene groups in fatty acid chains of the hepatic triglyceride will be evaluated 
with a line-fit procedure and commercial software (NUTS-ACORNNMR, CA).  
 
Blood sampling: Twelve-hour fasting blood samples will be collected at week 1, 13, and 24 consecutive 
feed and fast days (20 ml at each time point). All blood draws will be performed at the HNRU by Kelsey 
Gabel. Blood will be centrifuged for 15 min at 520 x g and 4°C to separate plasma from red cells, and will be 
stored at -80°C at the HNRU. Specimens will be labeled with the subject ID and study week, and will be 
stored until the study is completed. After the study is over, all specimens will be destroyed.  
 
Hepatic insulin sensitivity by OGTT and Matsuda Index: Hepatic insulin sensitivity will be assessed at week 
1, 13 and 24 by the oral glucose tolerance test (OGTT) and Matsuda Composite Index. 19 For this procedure, 
subjects will arrive at the research center in the morning after a 12-h fast. They will have their blood drawn 
(20 ml), and then be given a 75 g dose of glucose (Thermo Scientific Glucose Tolerance Test Beverage, 
Waltham, MA). Four blood samples (20 ml each) will be taken 30, 60, 90, and 120 min after the glucose 
dose for the measurement of glucose and insulin. Hepatic insulin sensitivity will be calculated as Matsuda’s 
Insulin sensitivity index (ISI)  = [10000/(fasting glucose x  fasting insulin x mean glucose during OGTT x mean 
insulin during OGTT)1/2]. 
 
Activity monitor: All subjects will be instructed to maintain their regular daily activity habits (i.e not join a 
gym or participate in other exercise classes). Alterations in physical activity habits will be quantified by an 
activity monitor (Fitbit Alta HR, Boston, MA)) that will be worn on the wrist for 7 days at week 1,13, and 24.  
 
Food record: Dietary intake will be assessed in all groups by a 7-day food record at week 1, 13 and 24. 
Subjects will be given detailed guidelines on how to complete the records by the Dietician at baseline. 
Dietary intake data will be assessed by Nutritionist Pro software (Axxya, TX).   
 
Michigan Alcohol Screening test: Alcohol consumption will be assessed by the Michigan Alcohol Screening 
Test at the screening appointment. 15 
 

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Metabolic disease risk factors: Plasma metabolic disease risk factors will be assessed at baseline week 
1,13, and 24. Plasma total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride concentrations will 
be measured in duplicate using enzymatic kits, standardized reagents, and standards (Biovision Inc, 
Mountainview, CA) and analyzed using a microplate reader (iMark Microplate Reader; Bio-Rad Laboratories 
Inc, Richmond, CA). HbA1c will be assessed by ELISA (Mybiosource, San Diego, CA). Fasting plasma glucose 
concentrations will be measured with a hexokinase reagent kit in duplicate (A-gent glucose test, Abbott, 
South Pasadena, CA). Fasting insulin will be measured as total immunoreactive insulin (Coat-A-Count 
Insulin, Los Angeles, CA). Insulin resistance (IR) will be calculated by the HOMA (Homeostasis Model 
Assessment) method: [HOMA-IR = Fasting insulin (µlU/ml) × Fasting glucose (mg/dL) / 405].  
 
Proteomics and metabolomics: Plasma-derived proteomic and metabolomic outcomes will be measured 
using immunoassay and mass spectrometry, as described previously.20, 21 
 
Hepatokine plasma levels: Plasma hepatokines will be measured at week 1, 13, and 24. Commercially 
available ELISA kits will be used to measure concentrations of Fetuin-A, fetuin-B, FGF-21, SHBG, and RBP4, 
(R&D Systems, Minneapolis), and adropin and selenoprotein P (Mybiosource, San Diego, CA). All samples 
will be assessed in duplicate. 
 
Inflammation: Inflammatory markers will be measured at week 1, 13, and 24. C-reactive protein (CRP) will 
be measured in duplicate using Immulite 1000 High Sensitivity CRP kits (Diagnostic Products Corpor- ation, 
Los Angeles, CA). Adiponectin, leptin, TNF-a, and IL-6 will be measured using ELISA (R&D Systems, 
Minneapolis, MN).  
 
Sleep questionnaires: Sleep quality and duration will be measured by validated questionnaires at week 1, 
13, and 24. The severity of insomnia in the past week will be measured by the Insomnia severity index (ISI), 
which is a 7-item questionnaire. Each item is rated by a 5-point Likert scale (where 0 indicates no problem, 
and 4 indicates a very severe problem) yielding a total score of 0-28. The total score for the ISI is 
interpreted as follows: no clinically significant insomnia (0-7), sub-threshold insomnia (8-14), moderate 
severity insomnia (15-21), and severe insomnia (22-28). Sleep quality, timing and duration will be measured 
by the Pittsburgh Sleep Quality Index (PSQI). This 19-item self-report questionnaire measures total sleep 
quality in the past month, yielding a total score of 0-21. A PSQI total score >5 indicates poor sleep quality. 
The questionnaire also asks for usual bedtime, usual wake time, and hours of actual obtained sleep. Risk of 
obstructive sleep apnea will be estimated using the Berlin Questionnaire at baseline. 
 
7.0 Expected risk and benefits 
 
Risks: Blood draws: Blood drawing may cause temporary discomfort from the needle stick, bruising, and 
infection. A total of 18 blood samples (20 ml each) will be collected over 24 weeks = 360 ml total.  
Alternate day fasting: Alternate day fasting is generally well tolerated and has no harmful effects. Subjects 
may feel hungry, however, which is unpleasant. DXA scans: DXA scans will be performed to assess body 
composition (3 scans total). Subjects who participate in this study will receive a small amount of radiation 
from the DXA scanning. The amount is similar to that received in many standard x-ray procedures, but is 
considerably more than subjects would receive from natural daily exposure or in the normal course of 
treatment, and it carries at least a theoretical risk. Magnetic resonance spectroscopy (MRS): There are no 
known hazards to MRS examination other than the discomfort associated with confinement and remaining 
stationary for the duration of the study. Testing of all subjects will be terminated immediately should they 
become distressed. We will carefully screen out individuals who have an implanted electrical device (such 
as a cardiac pacemaker or a neurostimulator), or a certain type of metallic clip (i.e., an aneurysm clip in the 
brain). These devices can malfunction and cause harm to the patient in the magnet environment.  We will 
also carefully screen individuals who are claustrophobic to reduce any adverse events. Confidentiality: To 
protect subject identity, only code numbers (i.e. randomly generated numbers) will identify all laboratory 
specimens, evaluation forms, reports, and other records. Exercise program: Risks of participating in the 

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exercise program include: muscle/joint strains, sprains, or injury. If a subject is injured during the exercise 
session, they will be asked to stop exercising immediately and rest. The muscle/joint will be iced and 
elevated, and the subject will be sent home with a small ice pack and asked to ice/elevate the area for 20 
minutes every hour to alleviate pain and inflammation. The subject will refrain from participating in the 
exercise session until the pain has significantly improved. If the pain does not improve in 48 hours, it will be 
recommended that the subject follow-up with their doctor. A cardiac event may also occur as a result of 
participating in the exercise program. If this occurs, 911 will be immediately called by the research assistant 
who is supervising the exercise session and the subject will be treated by a medical professional.  
Risk of coercion: Some subjects will be recruited from the Hepatology Clinic at the University of Illinois 
Medical Center by Dr. Sean Koppe MD (Co-investigator). Thus, there is a risk that subjects may feel coerced 
by Dr. Koppe to participate in the research. To mitigate this, each subject will be clearly informed that they 
are not obligated to participate in this research study offered by Dr. Koppe. Subjects will also be informed 
that participating in the study is completely voluntary and they are free to not participate. They will also be 
told that their decision to not participate will not affect your clinical care now or in the future. 
 
Benefits: Subjects may lose weight as a result of treatment. Since weight loss has been shown to improve 
some metabolic disease risk factors, subjects partaking in the treatment may experience these benefits to 
their overall health. No benefits can be guaranteed, however. 
 
8.0 Data collection and management procedures 
 
Sources of data include body weight and body composition measurements, hepatic steatosis measurement 
by MRS, hepatic insulin sensitivity by OGTT, blood pressure measurements, biochemical analyses of blood 
draws, activity monitor measurements, and questionnaire responses. Questionnaires will be stored in 
locked filing cabinets at the HNRU. To protect subject identity, code numbers only will identify all 
laboratory specimens, evaluation forms, reports, and other records. Subject identities will not be used in 
any reports or publications resulting from this study. All patient records will be kept in locked files; code 
sheets linking the patient’s name to a patient identification number will be stored separately in a locked file 
cabinet. Only study personnel will have access to the files. Blood specimens will be stored at the HNRU in a 
locked -80C freezer. Specimens will not be labeled with any personal identifiers. Only the subject ID and 
study week will be on the label of the blood specimens. Code sheets linking the patient’s name to a patient 
identification number will be stored separately in a locked file cabinet. 
 
 
9.0 Data analysis 
 
Data will be analyzed by Sally Freels PhD (Biostatistician) and Krista Varady by SPSS software (SPSS v24). 
 
10.0 Quality control and quality assurance  
 
The PI will be responsible for the evaluation of data quality, and this will be done on a weekly basis by 
evaluating biochemical analyses output and reviewing questionnaire responses. 
 
11.0 Data and safety monitoring 
 
Assessment of Risk: The risk of significant adverse events to participants in this study is low.  During the 24-
week study, participants will undergo 18 blood draws, 3 DXA scans, 3 MRS scans, alternate day fasting and 
exercise.  
Anticipated Adverse Events: Blood drawing may cause temporary discomfort, bleeding or bruising at the 
needle site and in rare cases fainting and infection. As for the DXA scan, the radiation dose associated with 
DXA measurements is very low or even insignificant in comparison with background radiation levels. 
Subjects may feel hungry as a result of the ADF diet, which may be unpleasant. Other forms of 
psychological stress may result from the dietary restriction or the demands of participating in and 
complying with a study. The subjects will be clearly informed of the expectations of the study at their 

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screening appointment (e.g. dietary protocol, exercise intervention, time commitment, etc.), and will be 
free to withdraw from the study at any point. There are no known hazards to MRS examination other than 
the discomfort associated with confinement and remaining stationary for the duration of the study.  
Testing of all subjects will be terminated immediately should they become distressed.  We will carefully 
screen out individuals who have an implanted electrical device (such as a cardiac pacemaker or a 
neurostimulator), or a certain type of metallic clip (i.e., an aneurysm clip in the brain).  These devices can 
malfunction and cause harm to the patient in the magnet environment.  We will also carefully screen 
individuals who are claustrophobic to reduce any adverse events. Loss of confidentiality may also be a risk. 
To protect subject identity, only code numbers (i.e. randomly generated numbers) will identify all 
laboratory specimens, evaluation forms, reports, and other records. Risk of participating in the exercise 
program includes: muscle/joint strains, sprains, or injury. If a subject is injured during the exercise session, 
they will be asked to stop exercising immediately and rest. The muscle/joint will be iced and elevated, and 
the subject will be sent home with a small ice pack and asked to ice/elevate the area for 20 minutes every 
hour to alleviate pain and inflammation. A cardiac event may also occur as a result of participating in the 
exercise program. If this occurs, 911 will be immediately called by the research assistant who is supervising 
the exercise session and the subject will be treated by a medical professional.  
Adverse Event Grading Scale:  Because only minor adverse events related to blood drawing, DXA scanning, 
MRS scanning, alternate day fasting and exercise may occur, no grading scale is necessary. 
Reporting of Adverse Events:  Reporting of adverse events will follow requirements mandated by the 
University of Illinois Office for Protection of Research Subjects (OPRS) and the NIH. The OPRS will receive a 
verbal or e-mail report of any serious adverse event (SAE) that occurs during the conduct of the study 
within 48 hours.  This will be followed by a detailed written report within 10 working days. In addition, any 
incidents or problems involving the conduct of the study or patient participation, including problems with 
the recruitment and/or consent processes will be reported within 10 working days. The NIH Program 
Official will be provided with a detailed written report of fatal, life threatening SAEs and unanticipated 
problems (UP) within 7 calendar days of the event. The NIH Program Official will be notified of any UPs that 
pose greater risk of harm to study participants than was previously known or recognized within 30 calendar 
days of the event. 
Safety Monitoring Plan: Sterile procedures will be employed for all blood drawing procedures. Subjects will 
be provided with a 24-hour contact phone number to report any persistent problems. The OPRS will be 
notified of any adverse events as described above. 
Frequency of Safety Reviews:  Safety reviews will be conducted annually in conjunction with the protocol 
renewal to OPRS. 
Persons to Perform Safety Reviews: The PI will be responsible for reporting any adverse events and 
conducting the annual safety review of study data.  
 
12.0 Statistical considerations  
 
All continuous variables will be examined for distributions and the presence of outliers. Variables that are 
not normally distributed will be transformed and if normality cannot be achieved, will be analyzed using 
non-parametric tests. Standardized descriptive statistics including measures of means, median, standard 
deviations, ranges, and standard errors for continuous variables within each group will be calculated to 
describe the groups at fixed time points. Differences between groups at baseline will be assessed by a one-
way ANOVA. A repeated measures ANOVA will be used to evaluate the effects of diet, exercise, and their 
interaction (diet x exercise) on dependent variables; a significant interaction will be interpreted with a 
Bonferroni correction for multiple comparisons. A multivariate analysis that adjusts for the effects of 
confounding variables (age, sex, race, and baseline hepatic steatosis, hepatic insulin sensitivity, hepatokine 
profile, body weight, and body composition) will also be performed. Relations between continuous 
variables will be assessed by Pearson’s or Spearman’s correlation coefficients as appropriate. Data will be 
analyzed using SPSS software (SPSS, Chicago, IL). 
 

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13.0 Regulatory requirements 
 
13.1 Informed consent: Consent will be obtained prior to the administration of the screening questionnaire 
by a member of the study team (Kelsey Gabel or key research personnel identified in Appendix P). The 
informed consent document will be stored in a locked filing cabinet at the Human Nutrition Research 
Center (1919 W Taylor St, Room 121C). All study team members have completed the CITI and HIPAA 
training courses. 
 
13.2 Subject confidentiality: To protect subject identity, code numbers only will identify all laboratory 
specimens, evaluation forms, reports, and other records. Subject identities will not be used in any reports 
or publications resulting from this study. All patient records will be kept in locked files; code sheets linking 
the patient’s name to a patient identification number will be stored separately in a locked file cabinet. Only 
study personnel will have access to the files. 
 
13.3 Unanticipated problems: Heart disease, diabetes and fatty liver disease risk parameters will be 
measured during the study. If the lab tests indicate that disease risk has increased as a result of 
participating in the study by week 12 or 24, the subject will be immediately informed. A printout of the lab 
values will be given to the subject and they will be asked to follow up with their doctor. Reporting of 
adverse events will follow requirements mandated by the University of Illinois Office for Protection of 
Research Subjects (OPRS) and the NIH. The OPRS will receive a verbal or e-mail report of any serious 
adverse event (SAE) that occurs during the conduct of the study within 48 hours.  This will be followed by a 
detailed written report within 10 working days. In addition, any incidents or problems involving the conduct 
of the study or patient participation, including problems with the recruitment and/or consent processes 
will be reported within 10 working days. The NIH Program Official will be provided with a detailed written 
report of fatal, life threatening SAEs and unanticipated problems (UP) within 7 calendar days of the event. 
The NIH Program Official will be notified of any UPs that pose greater risk of harm to study participants 
than was previously known or recognized within 30 calendar days of the event. 
  
14.0 References 
 
1. 
Lazo M, Hernaez R, Eberhardt MS, et al. Prevalence of nonalcoholic fatty liver disease in the United States: 
the Third National Health and Nutrition Examination Survey, 1988-1994. Am J Epidemiol 2013;178:38-45. 
2. 
Le MH, Devaki P, Ha NB, et al. Prevalence of non-alcoholic fatty liver disease and risk factors for advanced 
fibrosis and mortality in the United States. PLoS One 2017;12:e0173499. 
3. 
Bugianesi E, Moscatiello S, Ciaravella MF, Marchesini G. Insulin resistance in nonalcoholic fatty liver 
disease. Curr Pharm Des 2010;16:1941-51. 
4. 
Gaggini M, Morelli M, Buzzigoli E, DeFronzo RA, Bugianesi E, Gastaldelli A. Non-alcoholic fatty liver disease 
(NAFLD) and its connection with insulin resistance, dyslipidemia, atherosclerosis and coronary heart 
disease. Nutrients 2013;5:1544-60. 
5. 
Bell LN, Wang J, Muralidharan S, et al. Relationship between adipose tissue insulin resistance and liver 
histology in nonalcoholic steatohepatitis: a pioglitazone versus vitamin E versus placebo for the treatment 
of nondiabetic patients with nonalcoholic steatohepatitis trial follow-up study. Hepatology 2012;56:1311-8. 
6. 
Musso G, Gambino R, Cassader M, Pagano G. A meta-analysis of randomized trials for the treatment of 
nonalcoholic fatty liver disease. Hepatology 2010;52:79-104. 
7. 
Goodpaster BH, Delany JP, Otto AD, et al. Effects of diet and physical activity interventions on weight loss 
and cardiometabolic risk factors in severely obese adults: a randomized trial. JAMA 2010;304:1795-802. 
8. 
Shah K, Stufflebam A, Hilton TN, Sinacore DR, Klein S, Villareal DT. Diet and exercise interventions reduce 
intrahepatic fat content and improve insulin sensitivity in obese older adults. Obesity (Silver Spring) 
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9. 
Baumeier C, Kaiser D, Heeren J, et al. Caloric restriction and intermittent fasting alter hepatic lipid droplet 
proteome and diacylglycerol species and prevent diabetes in NZO mice. Biochim Biophys Acta 
2015;1851:566-76. 
10. 
Harvie M, Wright C, Pegington M, et al. The effect of intermittent energy and carbohydrate restriction v. 
daily energy restriction on weight loss and metabolic disease risk markers in overweight women. Br J Nutr 
2013;110:1534-47. 
11. 
Harvie MN, Pegington M, Mattson MP, et al. The effects of intermittent or continuous energy restriction on 
weight loss and metabolic disease risk markers: a randomized trial in young overweight women. Int J Obes 
(Lond) 2011;35:714-27. 
12. 
Meex RCR, Watt MJ. Hepatokines: linking nonalcoholic fatty liver disease and insulin resistance. Nat Rev 
Endocrinol 2017;13:509-20. 
13. 
Diabetes Management Guidelines. American Diabetes Association. 2017. Available from: 
http://www.ndei.org/ADA-diabetes-management-guidelines-diagnosis-A1C-testing.aspx.html. 
14. 
Pate RR, O'Neill JR, Lobelo F. The evolving definition of "sedentary". Exerc Sport Sci Rev 2008;36:173-8. 
15. 
Connor JP, Grier M, Feeney GF, Young RM. The validity of the Brief Michigan Alcohol Screening Test 
(bMAST) as a problem drinking severity measure. J Stud Alcohol Drugs 2007;68:771-79. 
16. 
Edition. ACoSMt. ACSM's Guidelines for Exercise Testing and Prescription. : Lippincott Williams and 
Wilkins.; 2017. 
17. 
Fealy CE, Haus JM, Solomon TP, et al. Short-term exercise reduces markers of hepatocyte apoptosis in 
nonalcoholic fatty liver disease. J Appl Physiol (1985) 2012;113:1-6. 
18. 
Haus JM, Solomon TP, Kelly KR, et al. Improved hepatic lipid composition following short-term exercise in 
nonalcoholic fatty liver disease. J Clin Endocrinol Metab 2013;98:E1181-8. 
19. 
Selimoglu H, Duran C, Kiyici S, et al. Comparison of composite whole body insulin sensitivity index derived 
from mixed meal test and oral glucose tolerance test in insulin resistant obese subjects. Endocrine 
2009;36:299-304. 
20. 
Perry A, Tanriverdi K, Risitano A, et al. The inflammatory proteome, obesity, and medical weight loss and 
regain in humans. Obesity (Silver Spring). 2023;31:150–158.  
21. 
Murthy V, Reis J, Pico AR, et al. Comprehensive metabolic phenotyping refines cardiovascular risk in young 
adults. Circulation. 202;142:210.