Effects of High-Intensity Interval Training Exercise in Adolescents With Hepatosteatosis
Phase: NA
Conditions: Non-Alcoholic Fatty Liver Disease, Insulin Resistance, High Intensity Interval Training, Childhood Obesity
Sponsor: Arkansas Children's Hospital Research Institute

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 1 
 
Study Title:  
Effects of High-Intensity Interval Training Exercise in Adolescents 
with Hepatosteatosis 
 
Principal Investigator:  
 
Emir Tas, MD 
Assistant Professor of Pediatrics 
Arkansas Children’s Hospital, Endocrinology and Diabetes  
1 Children’s Way, Slot 512-6, Little Rock, AR 72202 
Telephone: 501-364-1430, E-mail:  etas@uams.edu  
 
 
Co-Investigators: 
 
Jonathan A. Dranoff, MD 
Jerome S. Levy Professor of Medicine 
Director, Division of Gastroenterology & Hepatology 
University of Arkansas for Medical Sciences, Little Rock, AR 72205 
Telephone:  501-686-5545, E-mail:  dranoffjonathana@uams.edu 
 
Elisabet Borsheim, PhD  
Professor of Pediatrics  
Arkansas Children’s Nutrition Center, Arkansas Children’s Research Institute 
13 Children’s Way, Slot # 314, Little Rock, AR 72202  
Telephone:  501-364-3053, E-mail:  eborsheim@uams.edu  
 
Jon Oden, MD 
Associate Professor of Pediatrics 
Arkansas Children’s Hospital, Section Chief of Endocrinology and Diabetes   
1 Children’s Way, Slot # 512-6, Little Rock, AR 72202  
Telephone:  501-364-1430   E-mail:  jdoden@uams.edu 
 
Xiawei Ou, PhD 
Associate Professor of Radiology and Pediatrics 
Arkansas Children’s Hospital, Arkansas Children’s Nutrition Center   
13 Children’s Way, Little Rock, AR 72202  
Telephone:  501-364-4837   E-mail: ouxiawei@uams.edu 
 
Study locations: 
 
Arkansas Children’s (AC) 
Arkansas Children’s Hospital (ACH)  
Arkansas Children's Research Institute (ACRI) 
Arkansas Children's Nutrition Center (ACNC)  
 
 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 2 
Background and Rationale 
Non-Alcoholic Fatty Liver Disease (NAFLD) is characterized by fatty infiltration (steatosis ≥ 
5% of liver volume) and is the most common chronic liver disease of childhood. The estimated 
prevalence of NAFLD is 29% to 38% in obese children1. NAFLD is rare before puberty, but can 
be diagnosed during puberty. Routine screening for NAFLD is recommended in at-risk groups 
and is done via measurement of serum alanine aminotransferase (ALT) level, but this test has a 
low specificity1. Using Magnetic Resonance Imaging (MRI), a reference diagnostic test, we 
demonstrated that 40% of obese children (10-17 years) who attended to our weight 
management clinic had NAFLD. Twice the sex-specific ALT level, a recommended cut-point for 
further evaluation1, had only 30% specificity in our cohort. Further, ALT levels do not correlate 
with the stage of steatosis or fibrosis.2 Recent findings suggest that the degree of hepatic fat 
content (i.e., Intrahepatic triglyceride [IHTG] percent) in patients with NAFLD plays an 
independent role in progression of disease to advanced characterized by inflammation (Non-
Alcoholic Steatohepatitis [NASH]) and fibrosis3,4. This notion is supported by the finding that 
among adult NAFLD patients with no baseline fibrosis, those with a higher baseline IHTG 
(≥15.7%) developed fibrosis at a much higher rate (OR 6.67, 95% CI:1.01-44.1, p<0.05) after a 
1.75 year follow up5.  
Despite the high prevalence and associated comorbidities, patients with NAFLD are usually 
asymptomatic, and routine screenings are unlikely to identify the majority of the patients. Also, 
there are no FDA-approved medical therapies to treat NAFLD, and the current standard of care 
is limited to controlling risk factors via lifestyle modifications, which is more effective in earlier 
and reversible stages of the disease process. Affected individuals usually have cardiometabolic 
disorders.6,7 NAFLD is strongly associated with Insulin Resistance (IR), dyslipidemia, and 
hypertension, and is an independent risk factor for cardiovascular disease (CVD) morbidity and 
mortality8,9. In line with the literature, we found that all children with NAFLD in our cohort had IR 
(i.e., Homeostatic Model Assessment [HOMA]-lR ≥ 5) and lower cardiorespiratory fitness (as 
assessed by continuous treadmill graded protocol) and majority had markers of CVD 
(dyslipidemia or hypertension), but only 25% had elevated ALT (higher than twice the upper limit 
for sex). IR in the liver, muscle, and adipose tissue increases as IHTG increases10. Therefore, 
interventions aiming to decrease IR and improve cardiorespiratory risk factors will likely 
decrease IHTG, which may confer significant long-term health benefits. 
Clinical and Public Health Significance: Given unfavorable outcomes of youth-onset NAFLD 
compared to adult-onset, and increasing societal burden of the disease in parallel to rising 
childhood and adult obesity, every effort should be made at an early stage to mitigate the long 
term negative consequences of untreated disease. Unfortunately, adherence to lifestyle 
modifications is a real challenge for most adolescents.   
Scientific Premise: The mechanisms underlying NAFLD in adolescents with obesity have not 
been well-defined. Hepatocellular fat deposition is influenced by various factors, including 
circulating lipid levels, hepatic lipid uptake, de novo lipogenesis, hepatocellular fatty acid 
oxidation, and export of lipids from the liver11. Steatosis results from an imbalance between the 
forces/mechanisms favoring versus preventing intrahepatic fat deposition12.  IR is pivotal in the 
development of steatosis and progression to NASH1,11,13 According to the widely accepted 
adipose tissue (AT) expandability hypothesis14, lipids begin to accumulate in liver and other 
organs when the storage capacity of the AT is exceeded as a result of IR. An important premise 
and the empirical foundation for the current proposal rests on the relationships between NAFLD, 
IR, and cardiorespiratory fitness. Reversal of IR is associated with a decrease in IHTG and 
improvement of liver histology. In line with this, anti-diabetic mediations improve biochemical 
and/or histological findings in NAFLD, either directly through regulating hepatic lipid metabolism, 
or indirectly via increasing hepatic and peripheral insulin sensitivity15. In addition to IR, obesity is 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 3 
associated with lower cardiorespiratory fitness16, hypertension, increased heart rate variability17, 
atherogenic lipid profile18, and lower adiponectin levels. Obese subjects with NASH have even 
lower levels of adiponectin levels compared to obese with simple steatosis19. Supervised 
exercise, in as little as 4 weeks20,21, independently improves hepatic steatosis even in the 
absence of significant weight reduction or change in body composition, and increases 
adiponectin levels20,22–25. Additionally, exercise is thought to improve fibroblast growth factor-21 
(FGF21) signaling. FGF21 has been identified as a potential target for obesity and NAFLD 
treatment due to its strong association with both conditions. A multitude of studies show that 
FGF21 decreases IR, stimulates peripheral glucose uptake and lipid oxidation, and decreases 
lipogenesis, at least in part through increased adiponectin, and these effects are augmented via 
exercise26–29. 
Strategies aiming to decrease IR and increase fitness and adiponectin will likely reduce 
IHTG. Given the positive relationship between IR and steatosis and the inverse relationship 
between fitness, adiponectin, and steatosis, we propose that the exercise-induced reduction in 
IHTG will be more pronounced in those with a more significant decrease in IR  and increase in 
fitness and adiponectin. No previous pediatric studies have examined the longitudinal 
relationship between IR, fitness and adiponectin in response to exercise training to reduce IHTG 
in adolescents with NAFLD. The objectives of this study are to assess the cardiometabolic 
effects of a unique form of exercise strategy, high-intensity interval training (HIIT)30 on IHTG in 
adolescents with NAFLD and to examine and compare the relationship between IHTG, fitness, 
IR, and adiponectin. 
 Significance to Fundamental Science: In addition to clinical and public health significance, 
this project will begin to address how HIIT intervention, improved fitness and insulin action 
regulate IHTG metabolism, and provide novel insights as to the role of these factors in hepatic 
lipid homeostasis.   
Review of Relevant Literature: 
Decreased cardiorespiratory fitness is associated with NAFLD: Reduced fitness level is 
associated with NAFLD status and progression31. Higher baseline fitness, independent of body 
composition, is a predictor for the effectiveness of interventions for reducing IHTG in NAFLD 
patients32.   
HIIT decreases IHTG: HIIT, defined as alternating cycles of short periods of intense aerobic 
and anaerobic exercise at maximum capacity, increases aerobic and anaerobic fitness30. HIIT is 
a well-tolerated and efficient intervention that improves glucose metabolism and markers of 
CVD in overweight and obese children33–35. HIIT was effective in decreasing hepatic steatosis in 
adults with NAFLD24,31,36,37, but studies assessing the effect of HIIT on IHTG in obese 
adolescents with NAFLD are lacking. In a recent study involving mostly pre-pubertal children 
with NAFLD, Labayen et al.38 showed HIIT could effectively decrease IHTG, even when IHTG is 
marginally elevated (mean 5.6% in the treatment group).  
HIIT may directly regulate hepatic lipid metabolism in a weight independent manner: 
Mechanistically, HIIT stimulates hepatic lipid metabolism via hepatocyte transcription factor 
peroxisome proliferator-activated receptor alpha (PPAR-α)39,40 and AMP-activated protein 
kinase (AMPK)41,42. AMPK inhibits the synthesis of fatty acids, cholesterol, and triglycerides, and 
activates fatty acid uptake and oxidation. PPAR-α regulates beta-oxidation, mitochondrial 
function, and hepatic lipid metabolism31. Furthermore, PPAR-α is the upstream regulator for 
hepatic FGF21 expression43. Also, HIIT is associated with increased hepatocellular 
mitochondrial number and function40. These findings support the direct role of exercise on liver 
metabolism.  
HIIT improves cardiometabolic profile in obese individuals: HIIT  increases maximal oxygen 
uptake, decreases blood pressure and IR in overweight or obese children44 and adults45, and 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 4 
could be more effective in improving such risk factors compared to continuous, or low to 
moderate intensity interval training exercise46. A single bout of HIIT decreases insulin and 
glucose levels in healthy adolescents47. Obese adolescents with higher IR at baseline had the 
most decrease in IR following a total of 6 sessions of HIIT over 2 weeks.48 A multitude of adult 
studies49,50 show similar effects of HIIT on glucose level. HIIT decreases IR51–53 and increases 
adiponectin54,55 in obese adolescents.  
HIIT has a long-lasting positive effect on IHTG and cardiovascular risk factors:  Tjønna et 
al.56 demonstrated that 3 months of twice weekly HIIT in obese adolescents not only reduced 
CV risk factors immediately after the intervention, but also the effects were long-lasting. 
Specifically, fasting and 2-hour glucose, insulin, HOMA, blood pressure and V̇ O2max were all 
significantly different at 12-months post-intervention compared to baseline. Similar results, 
including reduction of IHTG, were observed in obese adults 12 months after the cessation of a 
one-year moderate to vigorous exercise intervention57, but these results were not replicated in 
another adult study which could be due to small sample size (n=10)58. Pediatric studies 
assessing the long term effects of exercise interventions on NAFLD are lacking. 
Objective/Specific Aims 
Based on available literature and our pilot data (shown below), we hypothesize that insulin 
resistance (IR) and decreased cardiac fitness are central to the NAFLD in adolescents, 
and the degree of steatosis is negatively correlated with lower fitness and higher IR. 
Thus, exercise-driven improvements in IHTG levels will be positively associated with the 
increase in fitness and the decrease in IR levels in adolescents with NAFLD. We will test 
our hypotheses through the following specific aims:  
Aim 1: Assess the change in IHTG, and CVD and IR markers in subjects with NAFLD after 4 
weeks of HIIT, compared to a sex-matched control group also with NAFLD with no training.  
1A. Quantitate the change in IHTG. Hypothesis: IHTG will decrease after HIIT, and the 
change will be greater than that seen in controls.  
1B. Measure the changes in fitness and markers of CVD. Hypothesis: HIIT will significantly 
increase fitness, decrease heart rate variability and blood pressure, and improve 
atherogenic lipid profile. The changes will be greater than that seen in controls. Changes in 
fitness and IHTG will correlate.  
1C. Estimate changes on IR. Hypothesis: HIIT will decrease IR especially in those with 
higher baseline IR, and the changes will be greater than that seen in controls. Changes in IR  
will correlate with changes in fitness and IHTG.  
Aim 2: Determine the post-intervention effects of HIIT on IHTG, and CVD and IR markers 3 
months after completion of the HIIT in adolescents with baseline NAFLD. 
2A. Quantitate the change in IHTG percent. Hypothesis: HIIT will have a sustained positive 
effect on IHTG, and that IHTG 3-months post-HIIT will be significantly lower compared to 
baseline level. 
2B. Estimate changes on IR and other CVD markers. Hypothesis: HIIT will have a sustained 
positive effect on IR, and that IR 3-months post-HIIT will be significantly lower compared to 
baseline level.  
 
Pediatric NAFLD has serious cardio-metabolic health outcomes. Findings of this study will 
inform clinical providers in the use of HIIT as a modality to treat adolescent NAFLD and 
associated morbidities, and produce preliminary data for mechanistic studies to probe pathways 
implicated in the pathogenesis of NAFLD. 
 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 5 
Preliminary Results  
We have successfully recruited 59 obese 
children for a pilot study and characterized 
their metabolic profile, including IHTG (via 
MRI)59. Forty percent of children had NAFLD 
in this cohort. Children with NAFLD have 
decreased 
cardiorespiratory 
fitness: 
We 
assessed fitness using continuous treadmill 
graded 
protocol60. 
While 
most 
healthy 
individuals are expected to walk 9-12 
minutes, the duration was only 4.5  3 min 
(range 1-7 min) in our cohort (either due to 
exertion or target heart rate been achieved). Elevated blood pressure (BP) was more prevalent 
in NAFLD group: Sixty percent of children with NAFLD had elevated BP z-score (adjusted for 
age, height, sex) compared to 33% of children without NAFLD. Children with NAFLD have 
higher IR: While all subjects had elevated IR as assessed by HOMA-IR, children with NAFLD 
had even higher HOMA-IR levels. Also, adiponectin (as an indirect marker for IR) was lower , 
and FGF21 was higher (likely a compensatory response) in 
NAFLD group (Table 1). IR and elevated FGF21 have an 
additive effect on IHTG: When all children were stratified 
based on their median HOMA-IR and FGF21 levels, those 
with HOMA-IR ≥5.67 and FGF21 ≥145.2 pg/mL had higher 
IHTG percent compared to other groups (Figure 1). 
Association of FGF21 and IHTG was dependent on the 
magnitude of IR. HOMA-IR values of the last two groups 
were comparable (9.74 ± 3. 65 vs. 9.18 ± 4.56, p=0.71). 
Taken together, our results and published literature11,61–63 
suggest that IHTG relates to insulin, FGF21, and 
adiponectin actions collectively. Strategies aiming to 
decrease IR and improve FGF21 sensitivity while 
increasing adiponectin levels could reduce  IHTG and treat NAFLD.   
Study Design and Procedures  
We propose a 4-week HIIT intervention in adolescents (ages 13 to 18, inclusive) with 
NAFLD. Up to 150 adolescents at-risk for NAFLD will be recruited to be able identify enough 
number of subjects with NAFLD (prevalence of NAFLD in our cohort was 40%; see data 
analysis section below). Our goal is to have 46 subjects with NAFLD to complete the study. 
Subjects will be randomized to either an exercise group or control group at a 5:1 ratio.  
Figure 1: IHTG per median HOMA-IR and FGF21 in 
obese adolescents (n=58). 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 6 
Because the objective of the study is to assess the effect of exercise in hepatic steatosis, we 
will use a multi-step approach to identify subjects with NAFLD and without disqualifiers (see 
exclusion criteria). When available, electronic medical records will be reviewed to identify 
potential participants. Eligibility will be assessed using a screening form. Potential participants 
may be screened over the phone or in person. If determined eligible, potential participants will 
be invited to attend a study visit. Study visits will take place at the Arkansas Children’s Nutrition 
Center (ACNC) and/or Arkansas Children’s Hospital. Parents and/or participants will be 
instructed not to come to the study visit and other assessments if parent and/or participant is ill. 
The visit/assessments will be rescheduled if that is case to a time when parents and participants 
are well and asymptomatic. Figure 2 summarizes the study visit schedule for the exercise and 
control groups as described in detail below.  
Participants will be asked not to eat or drink anything (except water) after 10 pm the night prior 
to the study visits. Due to fasting, study visits will be scheduled in the morning. Study 
compensation will be provided via ClinCard and/or gift card(s). Lunch and/or snacks may be 
offered to participants and/or parents at study visits.  
Baseline/Pre-Intervention: All Subjects  
Study Visit 1 – This visit may last up to 4 hours. During this study visit, participants will be 
further assessed for eligibility. We will perform the following measurements upon participant 
consent, or parental consent and participant assent: 
▪ 
Exercise Safety Assessment: Participants will be assessed for exercise safety using “The 
Physical Activity Readiness Questionnaire for Everyone” (2019 PAR-Q). Participants must 
be cleared for physical activity in order to continue in the study. If a participant is determined 
ineligible during the exercise safety assessment, study participation will be terminated 
before proceeding to the next measures. 
▪ 
Medical History & Pubertal Assessment: A medical history questionnaire will be 
administered to further confirm eligibility. In addition, because the participant’s pubertal 
development may influence the results from the imaging/blood samples, pubertal 
development must be taken into consideration. Subjects in the late stages of puberty will be 
recruited to minimize the confounding effect of puberty-associated changes in the body 
composition and insulin resistance on the test results. Confirming the pubertal status is 
essential because it is rare for a child to have NAFLD before puberty begins. Female 
participants will answer questions about their menstrual cycle. While males will be assessed 
by the study physician. The study physician will perform the pubertal assessment via Tanner 
Figure 2. Study Design. FMD, Flow-Mediated Dilation; DXA, Dual X-Ray Absorptiometry; OGTT, Oral Glucose Tolerance Test; MRI, 
Magnetic Resonance Imaging; ACNC, Arkansas Children’s Nutrition Center; HIIT, High-Intensity Interval Training. *These 
assessments will only be completed if Visit 5 takes place at ACNC. Note: Physical Activity (PA) assessment via FitBit will also be 
conducted throughout the study. 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 7 
staging using Prader Orchidometer, if the subject does not demonstrate clear signs of late 
stages of puberty (e.g., facial hair growth, deepening of the voice, completed linear growth 
as assessed in the growth chart). A testicular size of 12-15 mL or larger corresponds to 
Tanner stage IV in males.64 Pubertal assessment by self-report, particularly in males, is not 
a reliable measure of exact pubertal staging.65 Late stage of puberty (i.e., Tanner stage IV or 
V) must be confirmed in order to continue in the study. If a participant is determined 
ineligible during the medical history and/or pubertal assessment, study participation will be 
terminated before proceeding to the next measures.  
▪ 
Anthropometrics & Vital Signs: Body mass and height will be obtained using standardized 
techniques. In addition, we will also measure waist and hip circumferences using a tape 
measure. Blood pressure and heart rate will be assessed using standard techniques. If 
necessary, participants may be provided with a gown. Measures will be taken in duplicate, 
or triplicate if not within acceptable agreement.   
▪ 
FibroScan: A FibroScan (FibroScan® 530 Compact, Echosens, Paris, France) will be 
performed to determine NAFLD status based on Controlled Attenuated Parameter (CAP) 
score. FibroScan, similar to an ultrasound scan, is a technique to assess liver health. 
FibroScan is a routinely used clinical tool in most centers to assess liver health. Outcomes 
of FibroScan are “Controlled Attenuated Parameter (CAP)” and “Transient Elastogram (TE)”. 
CAP measurement strongly correlates with steatosis, while TE measures the stiffness of the 
liver as a surrogate marker of fibrosis, as described above. CAP values range from 100 to 
400 dB/m, and higher numbers indicate more pronounced steatosis. TE values range from 
2.5 kPa to 75 kPa, and higher numbers indicate more pronounced stiffness (fibrosis).  
Measurement via FibroScan is done when the subject is lying down on his/her back. The 
test involves a probe being passed over the right upper quadrant of the abdomen to 
measure the liver fat content and stiffness. The results are then immediately available for 
review once the scan is complete. No special preparation is required before the FibroScan. 
We will use the CAP cut-point of 241 dB/m as suggested by Shin et al. to determine 
baseline steatosis.66 A subject with a CAP score of 241 dB/m or greater will be accepted as 
having steatosis. FibroScan test takes about 20 minutes to complete including the 
generation and interpretation of the results.  
In the rare instance that a valid scan is not feasible, the participant will be accepted as 
having NAFLD and will be allowed to proceed with the study. If upon initial Magnetic 
Resonance Imaging (MRI), the participant is found not to have NAFLD, then participation 
will be discontinued at that time.    
Out of the participants whose eligibility is confirmed:  
▪ 
Oral Glucose Tolerance Test (OGTT): Standardized 2-hour OGTT, as described by the 
American Diabetes Association, will be performed to assess the indices of insulin sensitivity 
and secretion. Venous blood sampling for the measurement of serum glucose, insulin, 
FGF21, adiponectin, and other analyses/metabolites of interest will be done at time 0 and 
120 minutes by an experiences phlebotomist or physician via needle stick and/or 
intravenous line. Subjects will ingest a standard OGTT glucose drink (1.75 gram/kg, max 75 
gram) within 5 minutes of blood sampling at time 0. HOMA-IR, QUICKI Index, and Matsuda 
Index (also known as insulin sensitivity index) will be calculated as previously described.67,68 
Up to a total of 40 mL of blood will be collected during OGTT. Glucose concentration will be 
measured using a standard calibrated point of care glucometer to determine the diabetes 
status instantly. Subjects with fasting glucose concentration ≥126 mg/dL or a 2-hour glucose 
concentration ≥200 mg/dL will be considered to have diabetes, an exclusion criterion. These 
subjects will be referred to the diabetes clinic for the management of their conditions and will 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 8 
not continue in the study. Participants with no diabetes (those with fasting glucose <126 
mg/dL AND 2-hour glucose <200 mg/dL) will continue in the study. 
▪ 
Urine Collection: Urine will be collected to assess outcomes of interest, such as hydration 
status and to conduct metabolomics analyses. 
▪ 
Dietary Questionnaire: The Block Food Frequency Questionnaire and/or Block Food 
Screeners will be used to assess dietary intake. The records will be analyzed using 
appropriate software to provide high quality, comprehensive, and complete nutrient data. 
Also, participants will be asked to follow their normal diet throughout the study enrollment. 
Participants will be compensated with $50 at the end of the visit. Partial visits, due to ineligibility 
or any other reason, will be compensated with $25.  
Study Visit 2 – This visit may last up to 3 hours to complete. During this visit, the following 
measurements will be performed: 
▪ 
Flow-Mediated Dilation: Endothelial Function will be measured using ultrasound (GE Vivid 7 
Ultrasound system with 5.0-13.0 mHz linear transducer, GE Healthcare, Chicago, IL, USA) 
to assess brachial artery flow-mediated dilation (FMD). This technology uses sound waves 
(ultrasound) to visualize the diameter of the brachial artery. A baseline image of the brachial 
artery will be recorded at rest. Three ECG electrodes will be placed on the torso of the 
subject, such that ultrasound measurements will be captured at identical times during the 
cardiac cycle (i.e. ECG-gating of ultrasound images). A blood pressure cuff will be placed 
around the lower arm (the opposite arm that was used for measuring blood pressure) and 
the cuff will be inflated to 50 mmHg above systolic pressure for 5 minutes, followed by rapid 
deflation of the cuff. The diameter of the brachial artery will be measured using edge 
detection software (Vascular Tools, Medical Imaging Applications, IA, USA) from ultrasound 
images captured during the R wave of the cardiac cycle (via ECG-gating) throughout the 5-
minute recording protocol. The advantage of using ultrasound for FMD measurements is 
that this is a painless imaging test that uses non-invasive sound waves for imaging. There 
may be some discomfort from the blood pressure cuff since it will be applied tightly on the 
forearm. If the measurement is uncomfortable for the participant, and he/she requests to 
stop, then the procedure will be immediately aborted. FMD measurements will be done 
primarily by a registered clinical exercise physiologist or doctor with expertise in this 
procedure. However, other research staff trained and certified by Dr. Keshari Thakali 
(Director of the vascular physiology laboratory at ACNC) may conduct this measurement.  
▪ 
Pregnancy Screening: A questionnaire will be used to assess pregnancy status in female 
participants prior to Dual X-Ray absorptiometry scan in order to ensure safety. Pregnancy 
testing via urine sampling will be conducted if parent/participant chooses this option.  
▪ 
Dual X-Ray absorptiometry (DXA) scan: DXA scan is one of the best-standardized methods 
to assess the body composition (total body fat, lean body mass, visceral adipose tissue) of 
an individual. Body composition assessment is an essential part of the study due to the 
effects of whole-body adiposity and body composition on insulin resistance and NAFLD.  
▪ 
Fitness Test: All subjects will undergo a cycle ergometer stress test to assess their fitness 
level and determine peak oxygen uptake (VO2). This test will be conducted primarily by an 
ACSM registered clinical exercise physiologist (with M.Sc. in exercise physiology) with 
expertise in this procedure. Other research staff that have been certified by Dr. Elisabet 
Borsheim (Director of the Physical Activity Core at ACNC) may also conduct this 
measurement. Briefly, after evaluating the participants at rest and during a warm-up period, 
work load will be gradually increased until volitional exhaustion. The participants will be told 
that they are free to stop the test at any time if they do not want to continue the test, but will 

Title:  Effects of High-Intensity Interval Training Exercise in Adolescents with Hepatosteatosis  
PI:     Emir Tas, MD 
 
Version #:  14 
Date:  06.16.2021  
Page 9 
be encouraged to perform maximally. During the entire test, breath will be sampled and the 
composition will be measured using a metabolic cart (Medgraphics Ultima PFX® system, 
MGC Diagnostics Corporation, St. Paul, MN, USA). From this, the O2 uptake and CO2 
excretion will be determined, and their respiratory exchange ratio (O2 uptake/CO2 expiration) 
will be assessed throughout the test. The participants will be required to wear an indirect 
calorimetry face mask for these measurements. The participants will be given ample time to 
become familiar with the equipment and face mask. A heart rate monitor (Zephyr Bluetooth 
Wireless Heart Rate Sensor, Medtronic, Boulder, CO) will be worn to measure the level of 
cardiovascular activity during the exercise test. Participants will be trained on how to assess 
their exertion levels, and ratings of self-perceived exertion will be assessed every two 
minutes using the children’s OMNI scale.69 This is a numerical scale from 0 to 10, with a 
score of 2 indicating “a little tired” and a score of 9 indicating “very, very tired,” with 
associated pictures to represent perceived physical effort. Determination of peak VO2 will 
take the following into consideration: a rating of perceived exertion ≥ 8 on the OMNI scale, 
and/or a peak heart rate ≥185 beats/min (or ≥85% of predicted peak heart rate), and/or a 
respiratory exchange ratio ≥ 1.1. The validity of the OMNI score will be evaluated by 
presence of visible signs of fatigue, including inability to maintain pedaling cadence. 
▪ 
Randomization: Participants will be randomized to the control or exercise group. We will 
employ a randomized block design with sex as the blocking factor. Participants who are 
randomized to the control group will not undergo any exercise training. If randomized to the 
exercise group, participants will be offered to tour the exercise facility for familiarization with 
location and personnel.  
Participants will be compensated with $25 at the end of the visit. A FitBit activity tracker will be 
provided for those who complete the study visit to account for differences in activity level at 
baseline and throughout the study. 
Exercise Intervention (High-Intensity Interval Training (HIIT)): Exercise Group Only 
HIIT Intervention – Exercise sessions will take place in the ACNC fitness facility. The session 
will begin with a 5-10 minute low-intensity warm-up, followed by ten 1-minute intervals using 
exercise equipment (such as elliptical machine and/or bike and/or treadmill) at a work rate that 
elicits 80-90% of the maximal heart rate determined during the VO2max test. The participants will 
have two minutes to recover between each effort and will be encouraged to continue to turn the 
“pedals” over or walk slowly between high-intensity efforts. To finish the session, the 
participants will perform a 5-10 minute cool-down at a low intensity. Duration and/or intensity 
may be adjusted, if needed. Each participant will be asked to attend 3 weekly training sessions 
for 4 weeks. A minimum compliance rate of 50% will be required to remain in the study (at least 
6 training sessions) by the end of the training period (4 weeks). Participants will be encouraged 
to do home exercises in between training sessions, but these will not be recorded other than via 
FitBit. All exercise sessions will be primarily directed and monitored by an exercise trainer who 
staffs the Core. However, an exercise physiologist may also conduct exercise training. The 
exercise trainer or exercise physiologist will monitor heart rate and OMNI score during sessions. 
Each HIIT exercise session is expected to last up to 1 hour. Compensation will be provided in 
increments of $10, $20, $30 and $40 at the end of the 3rd, 6th, 9th and 12th HIIT exercise 
sessions, respectively. Therefore, participants who attend all 12 HIIT sessions will receive a 
total of $100.  
Participants can bring a friend or family member to workout with them if they would like. A 
separate release will be signed by the friend or family member in order to workout at the ACNC 
fitness facility. 
5-Week/Post-Intervention: All Subjects 

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Study Visit 3 – This visit may last up to 3 hours and include: measurement of body mass and 
height, FMD, pregnancy screening (if female), DXA, and fitness test as described above. 
Participants who are in the control group will complete this visit 5 weeks ± 1.5 week after study 
visit 2. Participants will be reimbursed for their time with $25 at the end of the visit.  
Study Visit 4 – This visit may last up to 4 hours and include: measurement of body mass and 
height, vital signs, OGTT, FibroScan, urine collection, and dietary questionnaire as described 
above. Participants will be reimbursed for their time with $50 at the end of the visit. 
3-Month Follow-up: Exercise Group Only 
Study Visit 5 – Participants from the exercise group will be asked to attend a follow-up visit 3 
months ± 2 week after last HIIT session. This visit may last up to 4 hours and include: 
measurement of body mass and height, hip and waist circumference, vital signs, pregnancy 
screening (if female), DXA, FibroScan, FMD, OGTT, urine collection, and dietary questionnaire 
as described above. FMD, pregnancy screening (if female) and DXA will only be completed in 
the instance that this visit takes place at ACNC. Participants will be reimbursed for their time 
with $50 at the end of the visit. 
Magnetic Resonance Imaging (MRI)  
In addition to the study procedures described above, all participants will complete a MRI scan 
before the baseline/pre-intervention fitness test, and another MRI scan before the 5-week/post-
intervention OGTT.  Furthermore, participants in the exercise group will complete an additional 
MRI scan before the 3-month follow-up OGTT. The MRI scans will be done at the Arkansas 
Children’s Hospital Radiology Department for the determination of hepatic fat content via 
magnetic resonance imaging. Each visit for completion of a MRI scan will last up to 1.5 hours. 
Participants will be reimbursed for their time with $50 after each visit.  
Physical Activity Assessment 
Furthermore, physical activity (PA) will be assessed throughout the study via FitBit device. The 
participant’s activity information from his/her FitBit will be assessed and recorded. Multiple 
assessments may be made throughout study participation via checking the data through the 
website and/or mobile application. In the case of lack of adherence to FitBit wear (e.g., no data 
in an entire week, wearing FitBit less than half of the time, etc.), we may ask the participant to 
share physical activity information (e.g., step counts, activity minutes, etc.) from any other 
phone app that has been already installed on his/her phone. For instance, most, if not all, 
iPhone or Android devices have “Health” apps by default that automatically store physical 
activity data. 
Any of the study measurements described above may be re-attempted during the study 
visit(s) if needed and if the participant is willing. In addition, subjects may be asked to return for 
a "repeat visit" in order to repeat and/or complete any study procedure(s). This will happen if the 
study procedure(s) is unsuccessful, incomplete, or cannot be completed due to unforeseeable 
reasons during the study visit. An additional $25 will be provided at the end of the repeat visit 
after re-attempt and/or completion. 
Moreover, participants in the control group will be offered an opportunity to exercise at 
ACNC fitness facility after he/she completes the study. This will include one month of 
supervised exercise training (up to 12 sessions) within two months from when the participant 
completes this study. Participants can bring a friend or family member to workout with them if 
they would like. A separate release will be signed by the friend or family member in order to 
workout at the ACNC fitness facility.     

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Study Population 
Recruitment of up to 150 participants will be conducted using IRB approved advertisements. 
Participants will be recruited from a variety of different settings, including select pediatric 
outpatient clinics with a high volume of at-risk patients for NAFLD (e.g., Weight Management 
Clinic, Gastroenterology Clinic, Adolescent Clinic, General Pediatric Clinic). Advertisements 
may be distributed in the form of flyers, postcards, and letters by direct mail, at information 
booths or events, or to various locations for posting or distributing, including but not limited to 
outpatient clinics, healthcare offices, health fairs, schools/universities, grocery stores, 
supermarkets, recreational centers, retail stores, websites (ACH, ACHRI, ACNC, UAMS, and 
others as applicable) and churches. Print and digital ads may appear in newspapers, 
magazines, social media, websites, and circulars. On-hold phone messages, text messages, 
and radio/television ads may also be used. In addition, research staff may contact parents who 
expressed an interest in our studies or who previously agreed to be contacted regarding future 
studies. Research staff may also contact parents/participants who have signed up for the AC 
research registry.  
AC electronic medical records may also be reviewed by study team to identify potential 
participants, and patients/parents may be contacted and informed about this study. In addition, 
staff, investigators, or other health care providers at Arkansas Children’s may assist in 
identifying participants at clinic visits and ask if they would like to be contacted about research, 
or if they would be interested in answering some screening questions. If interested, contact 
information may be obtained directly from patient/parent or from AC electronic medical record or 
from healthcare provider, so that the study team member may follow up to inform parent/patient 
about the study and ask screening questions. Also, when applicable, patients/parents may be 
screened in person at the time of their visit.  
In all recruitment instances, the research team member and/or health care provider will educate 
the parents and potential participants about the study and if interested determine potential 
eligibility. A copy of the consent and assent may be provided prior to the study visit for 
information. Eligibility will be assessed through the screening process prior to scheduling a 
study visit and will be reviewed/confirmed at the time of the visit.  At the visit, participant’s 
consent or parental consent and participant’s assent will be obtained in person. 
Study staff may also recruit patients at Arkansas Children’s with MyChart accounts. Study staff 
may access the electronic medical records of AC patients and search for eligible participants. 
Once study staff determines that a patient may be eligible, staff will post the study information in 
their MyChart account under the available studies page. This page lists all studies the child may 
be eligible for that have chosen to recruit using MyChart. On this page, the parent or patient has 
the option to indicate if they are interested in the study or not. If they are interested, the MyChart 
system will notify study staff. Study staff will follow up with the interested patient (if 18) or 
patient’s parent to provide more information about the study. This may be done by 
providing/mailing a letter/flyer to the parent and/or patient or by directly contacting parents or 
patients via phone or email to let them know about the research opportunity. If the parent or 
patient indicates an interest in the study, study staff will proceed to determine potential eligibility 
and schedule a visit where informed consent/assent would be obtained in person. 
Participants may also be recruited among those who have previously participated in other 
studies (see below). Contact information of these subjects will be looked up from their AC 
electronic medical records. The research team may call and/or send IRB approved ads, texts, 
emails, and letters to inform participants about the new study. 

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▪ 
Fibroblast Growth Factor-21: An Adjunct Biomarker for Early Detection of Non-Alcoholic 
Fatty Liver Disease in Children (IRB Protocol: 206278): Eighteen subjects, between the 
ages of 10-17 years completed this study between November 2017 and January 2019, were 
found to have NAFLD via magnetic resonance imaging. Of these, participants for whom 
consent was obtained to be contacted for future studies and have their information or 
samples used for future research may be recruited.  
▪ 
Liver Assessment via FibroScan (IRB protocol: 260576): Participants will be recruited based 
on their FibroScan results (i.e. CAP score of 241 dB/m or greater). Of these, participants for 
whom consent was obtained for contact for future research, use of information obtained over 
the course of this study in future research studies related to liver and/or metabolic health, 
and use of information obtained over the course of this study for screening purposes for 
other studies related to liver and/or metabolic health, may be recruited. 
Information from the AC medical records regarding a participant’s contact information, 
demographics, and health, including medical history, medical diagnoses, medicines, puberty, 
date of birth, anthropometrics, vital signs, blood and imaging results, and other test results and 
labs may be collected upon consent/assent. This information may be collected, retrospectively 
(i.e., collection of information already present in the medical record at the time of enrollment) 
and prospectively (i.e., collection of new information that may become available after study 
enrollment), from the time of study enrollment up to the time that data analysis for this study is 
complete and results have been published (up to 3 years from enrollment).  
Inclusion Criteria for Initial Enrollment:  
▪ 
Ages 13-18 years (inclusive) for both sexes  
▪ 
Any ethnic/racial background 
▪ 
English speaking competence  
▪ 
Obesity (BMI≥ 95th percentile for age and sex)  
▪ 
Low risk to participate in an exercise program during initial phone screening  
▪ 
Late stages of puberty (i.e., Tanner stage IV or V) 
o Female participants should have attained menarche. By definition, onset of 
menstrual cycles corresponds to a minimum of Tanner stage IV breast 
development in females. A verbal confirmation of the attainment of menstrual 
cycles will be enough.  
o For male participants, a verbal confirmation of late puberty signs (e.g., facial hair 
growth, deepening of the voice, completed linear growth) will be enough for initial 
enrollment. However, pubertal assessment will be conducted by the study 
physician upon consent/assent to confirm late pubertal status (see above). 
Inclusion Criteria to Continue in the Study:  
▪ 
Cleared for physical activity 
▪ 
Presence of NAFLD as determined by FibroScan CAP score or MRI (see above)  
▪ 
Confirmed lack of diabetes as determined by OGTT (see above) 
▪ 
Confirmed late stages of puberty (i.e., Tanner stage IV or V) (see above) 
▪ 
Confirmed eligibility per medical history (see above) 
Exclusion criteria: 
▪ 
Pre-pubertal or early stages of puberty   
▪ 
Pregnancy (verbal assessment) 
▪ 
Confirmed lack of NAFLD in the past 6 months via biopsy, FibroScan or MRI   
▪ 
Presence of an implantable medical device or metal objects in the body (a 
contraindication for FibroScan) 

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▪ 
High-risk to participate in an exercise program as determined by “The Physical Activity 
Readiness Questionnaire for Everyone” (see above)  
▪ 
Unable to cooperate with study procedures and tests as determined by study PI, 
including genetic or physical conditions impacting mobility over the past year  
▪ 
Having known chronic illnesses/disorders that may independently affect study outcome 
measures: type 1 diabetes mellitus, T2D, neurologic (e.g., epilepsy), developmental 
(developmental delay, autism spectrum disorder), endocrine (Cushing’s, growth 
hormone deficiency), hepatic (other than NAFLD), autoimmune, cardiac and renal 
disorders  
▪ 
Taking any of the following medications that can affect study outcome at the time of 
enrollment: insulin, metformin or any other anti-diabetics, antipsychotics, oral steroids, 
and anabolic drugs (growth hormone replacement therapy, testosterone, and 
oxandrolone). The participant will not be excluded from the study if he/she begins them 
during the study unless recommended otherwise by the primary physician.  
▪ 
Adolescents who have a history of claustrophobia.  
▪ 
Adolescents who need sedation in order to complete MRI. 
▪ 
Adolescents determined ineligible by the PI or delegated staff. 
Risks and Benefits 
Subject Confidentiality: A risk to study participants is the potential for loss of confidentiality of 
study data. Measures to protect the confidentiality of study data will be implemented as 
described in the Data Handling and Recordkeeping section below. 
Blood Sampling and OGTT: There is a small risk that participants will encounter bruising and/or 
infection after having blood taken, however, the use of well-established blood draw techniques, 
and antiseptic procedures will ensure minimal risk. Participants may also experience sickness or 
dizziness. Oral glucose tolerance test is routinely used in the clinics to screen for diabetes in at-
risk individuals. There is no known risks of glucose drinks. If a subject is identified having 
diabetes via OGTT, appropriate referrals will be arranged and care will be provided without 
further delay.   
FibroScan: There are no risks to having a FibroScan done. It is not invasive and is painless. It is 
similar to an ultrasound scan. Pregnancy is not a contraindication for FibroScan; however, 
pregnancy-associated physiological changes in liver structure may alter the results of 
FibroScan.  
A TE score greater than 8.5 kPa was previously shown to be associated with advanced stages 
of fibrosis. If we identify any subject with a TE score of 8.5 kPa or greater, we will consult the 
results with Dr. Jonathan Dranoff, who is an experienced hepatologist at the UAMS GI clinic and 
the co-investigator of this study, for further guidance. 
Dual-Energy X-ray Absorptiometry (DXA): This procedure will be used to determine body 
composition, and it exposes subjects to a very low dose of radiation (x-rays). The dose is only 
slightly greater than normal background radiation levels and is far less than that received during 
a standard chest x-ray (DEXA: < 0.1 mrem, Chest x-ray: 25 mrem). Otherwise, there are no 
known risks associated with the performance of this procedure. Because of the use of radiation, 
this procedure may pose risk to an unborn child. 
Fitness Test and HIIT exercise: During the fitness test and HIIT exercise sessions, participants 
may experience discomfort associated with exercise which is normal. However, this and any 
other procedure can be aborted at any time at the request of the participant.  The United States 
Centers for Disease Control and Prevention (CDC) and The American Heart Association (AHA), 
recommend that children be physically active for at least 60 min per day with moderate- or 

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vigorous-intensity in their aerobic activity.70,71 These entities stress that vigorous-intensity 
aerobic activity should be included on at least 3 days per week. In the event of an adverse event 
or unanticipated problems, this will be reported to the study PI, the IRB, and the study sponsor 
in accordance with IRB Policy 10.2. 
Magnetic Resonance Imaging: MRI is a safe and painless test that uses a magnetic field and 
radio waves to produce detailed pictures of the liver and identify hepatic fat content. It does not 
give radiation. During the procedure, a participant needs to stay still as much as possible to 
prevent distortion of the images. Because the MRI scanner we use generates a strong magnetic 
field, people with metal implants (e.g., cochlear implants, cardiac pacemakers, certain prosthetic 
devices, etc.) are not eligible to participate in this study. Individuals with claustrophobia will be 
rejected as well because the risk of giving sedation outweighs the potential benefits from 
participating in this study. ACH Radiology Department is a very well-established site for imaging 
in children; therefore the risk is minimal.  
Flow-Mediated Dilation: The risks are no more than what happens in everyday life, or in a 
routine physical examination where blood pressure is measured. The procedure may be 
uncomfortable, because the blood pressure cuff will stay inflated tightly on the participant’s arm 
for 5 minutes, which is longer than in a typical visit to the doctor.  There may be some 
discomfort, like the hand feeling cold and feeling like it is “asleep” from the blood pressure cuff 
since it will be applied tightly on the forearm. 
Subjects may directly benefit from participating in this study. As stated above, exercise is one of 
the mainstays of the NAFLD treatment. Participants will receive supervised exercise training 
from an experienced exercise trainer, or exercise physiologist. The knowledge gained from the 
study will shed light on the effect of HIIT exercise on NAFLD outcomes in adolescents. Findings 
from this study may help designing individualized physical exercise regimen for the patients with 
NAFLD.   
Data Handling and Recordkeeping 
The principal investigator, co-investigators, and study staff will complete and maintain 
appropriate CITI training. All study subject material will be assigned a unique identifying code or 
number. The key to the code and participants’ study data will be kept in locked file(s), and/or 
secure UAMS and/or Arkansas Children’s maintained server(s) and/or database(s) (e.g., 
REDCap). All data and communications will be recorded in standardized paper and/or electronic 
case report forms. Data will be collected and managed using REDCap. The data integrity will be 
password protected. Only study investigators and staff will have access to the code and 
information that identifies the subject in this study.  
The Principal Investigator will carefully monitor study procedures to protect the safety of 
research subjects, the quality of the data and the integrity of the study. In addition, the PI and 
primary mentor (Jon Dranoff, MD) will review the data on a quarterly basis together. The 
information gained from this study that can be linked to a subject's identity will not be released 
to anyone outside of the study team other than the subject, subject’s parent, and the subject's 
physician (if applicable). The results of this study may be published in scientific journals and/or 
presented at scientific meetings and conferences without identifiers. All procedures will be 
performed by appropriately trained and credentialed personnel supervised by the PI.  
Coded samples and/or information may be shared for the purposes of this study (i.e. within the 
purview of this protocol) with local collaborators (e.g., Arkansas Children’s Nutrition Center, 
Arkansas Children’s Research Institute, University of Arkansas for Medical Sciences, Arkansas 
Children’s Hospital) and/or non-local collaborators. At the conclusion of the study, the data will 
be stored to inform future proposals, grants, protocols, and procedures. The key to the code will 

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not be destroyed while information and/or samples are kept. If consented, information and/or 
samples may be used in future studies related to liver and/or metabolic health.  
Specimen Handling and Storage 
Samples will be stored in freezers in the Arkansas Children’s Nutrition Center and/or Arkansas 
Children’s Research Institute. The freezers are constantly monitored to ensure that the proper 
temperature is maintained. Samples will be identified using the subject’s unique study acronym 
along with the date of sample collection and type of sample (e.g. EDTA plasma, serum). 
Remaining samples may be stored indefinitely or until used up for future analyses. If consented, 
samples may be used in future studies related liver and/or metabolic health. Participants and/or 
parents may contact the study’s principal investigator, if they choose to withdraw consent to use 
samples and/or information in future research. In this case, a written letter addressed to the 
principal investigator expressing their desire to withdraw their consent would be required. If the 
information and/or samples have been shared or if publication of results has occurred, then we 
may not be able to remove information and/or samples. 
Data Analysis 
Exploratory data analysis will be conducted prior to formal statistical analysis. We will examine 
the distributions of all key variables to check for data errors and ensure that modeling 
assumptions are not violated. Numerical summaries including means, standard deviations, 
medians and histogram graphical techniques will be used for continuous variables and 
frequencies will be computed for categorical variables. For both aims, the primary outcome is 
IHTG level measured by both FibroScan and MRI, and secondary outcomes are fitness level, 
and various cardiometabolic disease markers (i.e.; insulin, glucose, adiponectin, etc.). The 
immediate (Aim 1) and sustained (Aim 2) effect of HIIT will be measured by comparing the 
changes in outcomes between groups using a linear mixed-effect framework with a subject-level 
random intercept. Changes in the outcomes are defined as difference between follow up visits 
(5-week and 3-month) versus the baseline. The independent variables will be group 
(intervention vs control), visit time (baseline, 5-week, 3-month) and their interaction term. This 
model specification can test the effect of intervention as well as difference between visits, while 
correctly account for the within-subject correlation due to repeated visits. Post-hoc contrasts of 
visit time between 5-week and baseline will be used to test the hypotheses under Aim 1, while 
contrasts between 3-month and 5-week will be used to test the hypotheses under Aim 2.  
As an exploratory aim, diagnostic performance of FibroScan for the NAFLD adolescent will be 
determined by discriminating NAFLD status defined by FibroScan against that by MRI to 
calculate sensitivity, specificity, positive and negative predictive values. Additional agreement 
statistics such as concordance correlation coefficient and Bland-Altman plots will be provided for 
the continuous measurements of IHTG levels measured by both methods.  
In our pilot data, the IHTG levels were 11.5±5.4 percent at baseline for NAFLD subjects (N=23). 
With a pre-defined clinically significant change of 20% relative decrease from baseline, we 
expect the NAFLD subjects’ fat percent will be decreased to an average of 9.2±5.4 percent after 
HIIT intervention, which is an equivalent effect size of 0.43. In order to detect this effect size at 
80% power, we will need 38 NAFLD participants in the intervention group, therefore 8 NAFLD 
subjects in the control group. Accounting for a 20% dropout rate and false-negative screening 
from FibroScan, we propose to enroll 58 subjects with NAFLD. Therefore, we need to screen no 
less than 150 subjects at-risk for NAFLD at baseline, to be able to identify 58 subjects with 
NAFLD (NAFLD prevalence was 40% in our pilot study, see preliminary data section above).  
Ethical Considerations 

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This study will be conducted in accordance with all applicable government regulations and the 
University of Arkansas for Medical Sciences research policies and procedures.  This protocol 
and any amendments will be submitted and approved by the UAMS Institutional Review Board 
(IRB) to conduct the study.   
The formal consent of each adult subjects or legal representative of minor subjects and the 
assent of minor subjects, using the IRB-approved consent and assent forms, will be obtained 
before subject is submitted to any study procedure. Any subject who turns 18 years old during 
his/her participation in the study will be re-consented to provide his/her own consent. All 
subjects for this study will be provided with consent/assent forms describing this study and 
providing sufficient information in language suitable for subjects to make an informed decision 
about their participation in this study. The consent form will also be available in Spanish for 
Hispanic parents who do not have English speaking competence. In this instance, in-person or 
remote interpreter services will be available during the consent process. However, for the 
remainder of the study, only remote interpreter services will be available. Participants may also 
intermediate communications with their Hispanic parents during participation in the study.  
A copy of the consent/assent may be mailed or e-mailed to parent/participant for information 
prior to enrollment. The person obtaining consent/assent will thoroughly explain each element of 
the document and outline the risks, benefits, and requirements of the study. The consent 
process will take place in a quiet and private room, and subjects may take as much time as 
needed to make a decision about their participation. Participation privacy will be maintained and 
questions regarding participation will be answered. No coercion or undue influence will be used 
in the consent process. The consent form must be signed by the subject or legally authorized 
representative and the person obtaining the consent, and the assent form must be signed by the 
minor participant. A copy of the signed consent and assent forms will be given to the 
participant/parent, and the informed consent process will be documented in the research record. 
In addition, a signed copy of the consent form may be placed in the patients’ medical record. 
Parents/participants may also be contacted (e.g., phone, text, email, letter) during their 
participation in the study and/or in preparation for study visits, including prior to enrollment. 
During the study, parents and/or participants may withdraw study consent verbally. In this case, 
the subject will no longer participate in the study and no further information, data, and samples 
will be collected. However, we may still keep and use any information, data, and samples that 
have been already collected from parent and/or participant. 
During the consent/assent process, parents/participants will also be given the option to allow for 
contact for future research, and allow for use of information and samples obtained over the 
course of this study in future research studies related to liver and/or metabolic health. If agreed, 
information and specimens will be stored indefinitely (or until used up) for potential use in future 
research studies. De-identified samples and information may be shared for future research use 
(i.e. outside the purview of this protocol) with local researchers (e.g., Arkansas Children’s 
Nutrition Center, Arkansas Children’s Research Institute, University of Arkansas for Medical 
Sciences, Arkansas Children’s Hospital) and/or non-local researchers. In this case, samples 
may only be shared with researchers who who do not express a plan to use them for genetic 
research. In addition, inquiring researchers will be informed that the stored samples do not have 
consent for future genetic research. Participants may choose to withdraw permission for future 
use of information and/or samples at any time they decide. A written letter addressed to the 
principal investigator expressing their desire to withdraw their consent would be required. If the 
information and/or samples have been shared or if publication of results has occurred, then we 
may not be able to remove information and/or samples. Consent for future contact and use of 
information and/or samples for future research will be separate from consent to participate in the 

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current study. Their decision will not affect their eligibility for this study. Participants may call the 
investigators or research staff to obtain more information if needed. 
In addition, consent for release of data may be obtained from participants who have also taken 
part in Fibroblast Growth Factor-21: An Adjunct Biomarker for Early Detection of Non-Alcoholic 
Fatty Liver Disease in Children (IRB Protocol: 206278) and/or Liver Assessment via FibroScan 
(IRB protocol: 260576). If parent/participant agrees to release data, a copy of the signed 
consent for release of data will also be provided. 
Dissemination of Data 
Results of this study may be used 
for presentations, posters, publications, or 
intramural/extramural grant applications. The publications will not contain any identifiable 
information that could be linked to a participant. The study will be listed on clinicaltrials.gov in 
accordance with requirements.  
 

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