Break It Up: A Study Evaluating Breaking Up Daily Sedentary Behavior in Youth
Phase: PHASE2
Conditions: Healthy, Obesity, Overweight
Sponsor: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)

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Protocol Title:  Break It Up:  A study evaluating breaking up daily sedentary behavior in 
youth. 
Abbreviated Title:  Evaluation of interrupting daily sedentary behavior 
Protocol Number: 17-CH-0130 
Date of This Submission/Version: February 13, 2020 
Principal Investigator: 
Name, 
Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
Jack A. 
Yanovski, 
MD, PhD 
 
Section on 
Growth and 
Obesity, NICHD 
Building 10, 
Rm 2-3130 
301.496.0858 
jy15i@nih.gov 
 
Additional Investigators: Please see Study Personnel Sheet 
 
Study type (check all that apply): 
        x      Archived biological specimens/medical information 
                Natural history; definition of phenotype, genotype/phenotype correlation 
                Prospective linkage/gene identification, NOT providing information to participants 
                Prospective linkage/gene identification, providing information provided to participants  
                Social science; assessments of knowledge, attitudes and behavior 
                Genetic counseling 
                Drugs or devices 
                Gene transfer 
        x      Other interventions 
  
Estimated Duration of study: 2 years 
Human Research Protections Program Investigator and Staff Training: 
For this protocol, the following “Just in time” human subjects protection training courses are 
required for investigators and staff: 
• Biomedical- Vulnerable Subjects - Research with Children 
• CITI GCP modules 

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• Unanticipated Problems and Reporting Requirements in Biomedical Research 
• Unanticipated Problems and Reporting Requirements in Social and Behavioral Research 
 
Total requested accrual  
Subjects of Study: 
 
 
Number 
Sex 
 
Age Range  
Healthy Cohort (BMI 5th to < 85th percentile) 
--Screened but not enrolled 
 
15 
 
M, F 
 
7-11.99 y at study entry 
--Randomized  
 
 
60 
 
M, F   
7-11.99 y at study entry  
Overweight Cohort (BMI ≥ 85th percentile) 
--Screened but not enrolled 
 
15 
 
M, F 
 
7-11.99 y at study entry 
--Randomized  
 
 
60 
 
M, F   
7-11.99 y at study entry  
 
Project Uses Ionizing Radiation:  
 No   
Yes  
 
 Medically-indicated only 
 Research-related only (attach RSC/RDRC documentation) 
 
 Both (attach RSC/RDRC documentation) 
 
IND/IDE 
 
 
 
 No   
Yes (attach FDA documentation) 
  
Drug/Device/#_____________________ 
  
Sponsor: __________________________ 
 
Durable Power of Attorney   
 No   
 Yes  
 
Multi-institutional Project 
 
 No   
 Yes  
 
Data and Safety Monitoring Board   No   
 Yes  

3 
 
 
Technology Transfer Agreement  
 No   
 Yes  
 
Samples are being stored  
 
 No   
 Yes  
 
Flesch-Kincaid reading level of consent form: ____9.6________  
Flesch-Kincaid reading level of assent form: _______5.8_____ 
 

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Précis: 
 
Background:  Sedentary behavior is defined as a set of low-intensity activities involving limited 
body movement (e.g.: TV viewing, prolonged sitting). Some studies have found higher levels of 
childhood sedentary behavior predict higher body mass index (BMI) and metabolic 
abnormalities. We and others have found that interrupting sitting with short, 2-3 minute bouts of 
moderate activity (walking) can improve glucose tolerance during a single session. Thus, 
interrupting sedentary behavior may be an intervention strategy to reduce health risks. 
Objective:  We propose to conduct a randomized pilot study to assess whether interrupting 
sedentary behavior for 6 consecutive days provides sustained improvement in carbohydrate 
metabolism without negatively impacting executive function, attention, mood, anxiety, dietary 
intake or usual physical activity. Design & Population:  Using a randomized parallel group 
design, children, ages 7-11.99 years, will complete an assigned randomized condition of either 6 
consecutive days of 3 hours of monitored sedentary activity (sitting) or 6 consecutive days of 3 
hours of interrupted sitting (in which they will be prompted to walk for 3 minutes every 30 
minutes). Outcome measures: Twenty-four-hour continuous glucose monitoring and 
postprandial insulin incremental area under the curve (iAUC) on post-condition oral glucose 
tolerance testing will be the primary measures.  Secondary measures include:  postprandial 
glucose iAUC, executive function, attention, mood, anxiety, dietary intake, and free-living 
physical activity. Impact:  This project will investigate if consecutive daily interruption of 
sitting behaviors improves glucose tolerance, a potential negative health consequences of 
sedentary behavior in children. If repeatedly interrupting sitting with short bouts has sustained 
beneficial effects among children, interventions examining the frequency, duration, and intensity 
of such interruptions could be developed for use in the community setting. Thus, these results 
have the potential to provide insight into novel behavioral intervention targets in youth. 

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II. Table of Contents 
Outline/Table of Contents: 
 
 
 
 
 
 
 
       Page 
Précis 
 
4 
List of Abbreviations  
 
6 
1. 
Introduction and Background 
8 
 
a. 
Obesity and Sedentary Behaviors 
8 
 
b. 
Effects of Interrupting Sedentary Behavior 
11 
 
c. 
Continuous Glucose Monitoring 
15 
 
d. 
Executive function and Attention 
18 
 
e. 
Conclusion 
22 
2. 
Study Objectives and Specific Hypotheses 
24 
3. 
Subjects 
25 
a. 
Description of study populations 
25 
 
i. 
Inclusion Criteria 
26 
 
 
ii. 
Exclusion Criteria 
26 
4. 
Study Design and Methods 
28 
a. 
Study Overview 
28 
 
i. 
Trial Design 
28 
 
ii. 
Visit Work Flow 
29 
 
iii. 
Relationship with other protocols 
29 
b. 
Recruitment 
 
29 
i. 
Description of Recruitment Strategy 
29 
ii. 
Recruitment Methods 
30 
iii. 
Anticipated Accrual Rate 
30 
iv. 
Initial contact 
30 
 
c. 
Screening 
 
30 
 
d. 
Study Procedures 
36 
i. 
Test Days #1-6 Daily Procedures 
36 
ii. 
Test Day #1 
39 
iii. 
Test Day #5 
40 
iv. 
Test Day #6 
40 
v. 
End of Participation 
43 
5. 
Management of Data and Samples 
44 
 
a. 
Storage 
 
44 
 
b. 
Data and Sample Sharing Plan 
45 
 
c. 
Collaborating Researchers and Affiliated Institutions 
47 
6. 
Additional Considerations 
47 
7. 
Risks and Discomforts 
47 
8. 
Subject Safety Monitoring 
50 
 
a. 
Criteria for withdrawal of subjects from study 
50 
9.  
Outcome Measures 
 
51 
a. 
Primary Outcome Measure 
51 
b. 
Secondary outcomes  
51 
10. 
Statistical Analysis 
 
52 

6 
 
a. 
Analysis of Data 
52 
b. 
Power Analysis  
53 
11. Human Participant Protections 
54 
a. 
Rationale for Subject Selection 
54 
b. 
Equitability 
 
54 
c. 
Justifications for exclusions 
54 
d. 
Justification for sensitive procedures 
55 
e. 
Safeguards for vulnerable populations (Children) 
55 
f. 
Qualifications of Investigators 
Error! 
Bookmark not defined. 
12. 
Risks/Benefits Analysis 
55 
a. 
Anticipated Benefit 
Error! 
Bookmark not defined. 
b. 
Classification of Risk (for the study as a whole 
Error! 
Bookmark not defined. 
c. 
Assessment of Risk/Benefit Ratio 
Error! 
Bookmark not defined. 
13. 
Consent/Assent Documents and Process 
56 
a. 
Designation of those obtaining consent  
56 
b. 
Consent procedures 
56 
c.  
Non-English–Speaking Participants 
57 
d. 
Consent documents 
58 
14. 
Data and Safety Monitoring 
58 
 
a. 
Data and Safety Monitor 
58 
 
b, 
Data and Safety Monitoring Plan for Non-IND 
58 
15. 
Quality Assurance 
 
60 
16. 
Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations 60 
17. 
Alternatives to Participation 
61 
18. 
Privacy 
 
 
61 
19. 
Confidentiality 
 
61 
20. 
Conflict of Interest 
 
 
62 
21. 
Technology Transfer   
 
62 
22. 
Research and Travel Compensation  
 
62 
23. 
References 
 
 
 
64 
Appendix A1: Public Media Announcement 
Appendix A2:  Recruitment Mailing Letter 
Appendix A3:  Patient Flyer 
Appendix A4:  Listserv Email Template 
Appendix B:  Study Visit Flow Table 1 
Appendix C:  Protocol Consent 
Appendix D:  Protocol Assent 
Appendix E:  Inclusion and Exclusion Checklist [E.1 Supplemental MRS Arm] 
Appendix F:  Data Sharing Planned, Approval from Scientific Director 

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Appendix G:  Questionnaires (Hollingshead, IPAQ, Daily Health History) 
Appendix G2:  Clinical Trials Survey System Questionnaires 
List of Abbreviations  
• 
BMI: Body mass index 
• 
CGM:  Continuous Glucose Monitor 
• IMCL:  Intramyocellular lipid content 
• 
MAGE:  Mean amplitude glycemic excursion 
• 
MET:  Metabolic equivalent of task 
• MRS:  Magnetic Resonance Spectroscopy 
 
 

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1. Introduction and Background 
 
Pediatric Obesity and Sedentary Behaviors 
Excessive weight gain in children is one of the foremost global health concerns because 
of its metabolic consequences.1, 2 In the United States, nearly 1 in 5 children who are 6-11 years 
old are considered obese (BMI ≥ 95th percentile) by the Centers for Disease Control and 
Prevention standards, almost quadrupling since 1971.3  Excessive weight gain in childhood is 
associated with many adverse outcomes. Children with excessive weight have increased 
incidence of impaired glucose metabolism, dyslipidemia and cardiovascular strain. 4, 5  Long 
term, such children demonstrate increased incidences of musculoskeletal complications6-8 and 
have more difficulty with chronic illnesses such as asthma9 and depression.10  These childhood 
morbidities are reason enough to demand investigation and intervention but childhood obesity is 
also predictive of adult obesity and with it numerous adult morbidities including cardiovascular 
disease, metabolic syndrome, cancer, and increased all-cause mortality.11, 12  
The single most important factor of pathologic weight gain is an unhealthy balance 
between energy intake and energy expenditure.  One component of energy expenditure that has 
received more attention in the last decade is sedentary behavior.13, 14  Sedentary behavior is 
defined as low-intensity [<1.5 metabolic equivalent of task (MET)] activity that involves limited 
body movement.14 Sedentary behavior encompasses TV viewing, computer use and prolonged 
sitting, and is highly prevalent in the modern society of developed countries.    
Adult Studies on Sedentary Behavior and Obesity 
There is a growing body of work describing the influence of sedentary behavior on health 
outcomes in adults. Field studies provide support for the role of sedentary behavior in poor 
health outcomes. In a classic study, Morris et al. observed that sedentary bus drivers had higher 
rates of cardiovascular disease (CVD) than the more active conductors, independent of waist 
circumference.15 Similar increased CVD risk is seen in patients with spinal cord injuries who are 
forced to spend the majority of the day seated.16 This is supported by a review of longitudinal 
studies that suggest a consistent positive relationship between sedentary behavior, mortality and 

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weight gain from childhood to adulthood.17 These findings support the negative health effects of 
sedentary behavior that may begin in childhood and increase the risk for disease in adulthood. 
Pediatric Studies on Sedentary Behavior & Obesity 
Using data from the National Health and Nutrition Examination Survey (NHANES), 
Matthews et al. estimated that U.S. 6-11 year olds spend over 6 hours per day in sedentary 
behaviors.18 Furthermore, a high amount of time spent in sedentary behaviors is associated with 
pediatric obesity.19-21  This is supported by the finding that sedentary behavior in youth predicts 
greater gain in BMI from 9 to 15 years22 independent of moderate to vigorous physical activity 
(MVPA). Summative data from a recent meta-analysis reported that nineteen longitudinal studies 
found greater time spent watching TV at baseline predicted greater increases in BMI and fat 
mass over time,23 and other reviews indicate that the majority of studies report positive 
associations between reported TV viewing and body fat mass.24-27  
Sedentary Behavior & Metabolic Dysfunction 
In addition to weight gain, sedentary behavior has been associated with metabolic 
dysfunction.  Extensive epidemiology and clinical observational data in adults associate negative 
metabolic outcomes and sedentary behavior.28-31   For example, Healy et al. evaluated markers of 
the cardiometabolic profile and accelerometer-derived activity in 4757 subjects >20 years in the 
US National Health & Nutrition Examination Survey (NHANES).31 This study identified 
persistent negative associations such that, while controlling for moderate to vigorous activity, 
increased sedentary time in adults was associated with worsening HDL-cholesterol, C-reactive 
protein, triglycerides, and the homeostatic model of insulin resistance.31  Additionally, Balkau et 
al. identified a significant association between sedentary time (also recorded with accelerometry) 
and insulin resistance as measured by hyperinsulinemic-euglycemic clamps in 801 adults ages 
30-60 years.28  This finding was only partially attenuated when adjusted for body fat.28   
However, data have been more contradictory in youth.  In 308 children ages 9-10 years 
old, accelerometer-measured sedentary behavior was significantly associated with insulin 
resistance as measured by the homeostatic model of insulin resistance independent of fat mass.32 
Conversely, using a large pooled convenience sample of 20,871 children ages 4-18 years 
supplied by the International Children’s Accelerometry Database, Ekelund et al. reported that 

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total sedentary time measured by accelerometry was not associated with cardiometabolic risk (as 
measured by waist circumference, systolic blood pressure, fasting triglycerides, high-density 
lipoprotein or insulin) after adjusting for MVPA.33 The conflicting findings among the studies 
may be related to age, as sedentary time is higher in adolescence than childhood,34 and may 
change the power to detect associations. The conflicting findings in pediatric studies may also be 
due to how sedentary behavior is measured. The majority of published studies operationalized 
sedentary behavior as TV viewing time. TV viewing has traditionally been assumed to be a good 
measure of sedentary behavior in children, yet it is weakly correlated (r < .20) with device-based 
measures (e.g.: accelerometers) of sedentary time,35, 36 suggesting that they are measuring 
different behaviors. These screen-based behaviors may be a marker for other behaviors 
associated with poor health or socioeconomic status that could drive relationships with 
adiposity.24 However, other self-report measures designed to capture multiple sedentary 
behaviors (e.g.: the 3-day physical activity recall) have found that sedentary behavior is 
associated with increased risk of metabolic disease.37  In-lab studies have the potential to greatly 
contribute to this area through the investigation of basic biological pathways between sedentary 
behavior and metabolic characteristics and clarify the existing literature. 
Thus, in summary, recent data suggest that sedentary behavior may be contributing to the 
perpetuation of the high prevalence of pediatric obesity and metabolic dysfunction. Diet and 
increasing physical activity have been largely unsuccessful at producing long-term effects on 
body fatness.38-40 There is a need for novel behavioral intervention strategies to prevent pediatric 
obesity and its metabolic complications. Interrupting sedentary behavior is an innovative 
technique. 
Intervention:  Limiting Sedentary Behavior 
Limiting sedentary behavior has been proposed as a strategy for pediatric obesity 
prevention and intervention. 1, 41-43 Sedentary behavior has most often been operationalized as 
TV viewing or screen-based behavior. In a seminal randomized controlled trial, Robinson et al 
reported that children who received an intervention aimed at reducing TV viewing had 
significant decreases in BMI, waist circumference, and waist-to-hip ratio.44  A recent review 
reported that interventions aimed at reducing TV viewing produce modest improvements in 
weight parameters and slowing of BMI increases during childhood and adolescence.45 However, 

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many of these interventions included other health messages, so it is difficult to distinguish the 
contributions of the TV-reduction messages alone. Also, continued research on the physiologic 
mechanisms linking sedentary behavior and obesity may help explain potential behavioral 
intervention targets that go beyond TV viewing.  
Effects of interrupting sedentary behavior 
As humans continue to spend the majority of their day sedentary,46 it is important to 
investigate how intervening on this behavior influences health outcomes. In adults, 
epidemiological and cross-sectional evidence suggests that interrupting prolonged sedentary 
behavior is associated with: lower waist circumference,31 lower triglyceride levels,47 and lower 
postprandial glucose.48 In children, school-based studies indicate that breaks in sitting increase 
attention.49 Reviews of the literature did not find sufficient support for the prospective50 or cross-
sectional51 effects of sedentary behavior on cardiometabolic health outcomes. However, the 
findings from the reviews must be interpreted cautiously because sedentary behavior was 
measured differently across the reviewed studies, and there were no randomized control trials 
that manipulated the behavior. 
Pathophysiologic basis: 
There are acute physiologic mechanisms that relate sedentary behavior with increased 
metabolic risk which support the potential effectiveness of interrupting sedentary behavior.52 
Early research in this area focused on the acute effects of prolonged sedentary behavior on 
lipoprotein lipase (LPL) activity as a potential link between behavior and health risk. LPL is 
central in lipid metabolism, and plays a role in free fatty acid (FFA) uptake among different non-
hepatic tissues.53 LPL concentration is high in oxidative (slow twitch, postural) muscle, and is 
hypothesized to mediate the relationship between sedentary behavior and cardiometabolic risk 
because prolonged sitting reduces the use of oxidative muscles (such as those used for standing), 
lowers LPL, and reduces the body’s ability to take up fatty acids into tissues after a meal. The 
result is prolonged exposure of the arterial walls to plasma triglycerides that increases 
cardiometabolic risk.54, 55 This mechanistic hypothesis may explain why cross-sectional pediatric 
studies have found that more time spent in sedentary behavior is associated with increased 
concentration of circulating cell adhesion molecules that play a role in the development of 
atherosclerosis and type 2 diabetes.35 Thus, sedentary behavior may exert a cumulative effect on 
health risk over the lifespan. 

12 
 
Sedentary behavior has also been associated with chronic physiologic alterations that 
may increase the risk for type 2 diabetes. Prolonged sedentary behavior is associated with lower 
insulin sensitivity and increased postprandial glucose concentrations.56 Sustained sedentary 
behavior diminishes the use of skeletal muscle to assist with glucose uptake from the 
bloodstream after a meal, requiring the pancreas to secrete more insulin that may increase the 
risk for beta cell dysfunction and type 2 diabetes.57, 58 Also, once insulin-mediated glucose 
uptake by skeletal muscle is decreased due to prolonged sitting, the elevated glucose 
concentrations lead to a longer exposure to high circulating insulin in the blood. This 
hyperinsulinemia promotes lipogenesis (fat synthesis) in the liver and uptake of fatty acids into 
adipose tissue.59 The result is increased adiposity and worsening insulin resistance.60 These 
pathways still need to be further explored to explain how the duration and patterns of sedentary 
behavior may produce negative health effects. 
 
Adult Studies Interrupting Sedentary Behavior 
To date, there have been a few 
published studies evaluating the 
effect of interrupting sedentary 
behavior on metabolism. 48, 61-63  
Dunstan et al. conducted a 
randomized crossover study where 
prolonged sitting was interrupted 
with 2 minutes of light- and 
moderate- intensity walking every 20 
minutes for five hours in 
overweight/obese adults (Figure 1).48 
They hypothesized that a lower 
postprandial glucose response would 
be observed when interrupting sitting 
with brief sessions of activity, 
regardless of the intensity of the 
Uninterrupted
Sitting + Light- Sitting + Moderate-
0
1
2
3
4
5
6
7
8
Sitting
Intensity
Activity Breaks
Intensity
Activity Breaks
p<0.01
Plasma Glucose iAUC
(mmol/L • hr)
Uninterrupted
Sitting + Light- Sitting + Moderate-
0
100
200
300
400
500
600
700
800
900
Sitting
Intensity
Activity Breaks
Intensity
Activity Breaks
p<0.01
Plasma Insulin iAUC
(pmol/L • hr)
 
Fig 1. Dunstan et al. Diab Care 2012;35:976-983 

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activity. Study participants attended three lab visits in random order to complete the three 
conditions: prolonged sitting, sitting interrupted by light walking, and sitting interrupted by 
moderate walking. A standard test drink was given at time 0 after which blood was taken every 
hour. Compared to the prolonged sitting condition, the glucose responses were significantly 
lower in the light- and moderate- intensity walking conditions – 24.1% and 29.6% respectively – 
with both conditions statistically significant vs. sitting (p<0.0001). Compared to the prolonged 
sitting condition, the insulin incremental area under the curve (iAUC) was 23% lower in the 
light- and moderate- intensity walking conditions. There were no significant differences in 
glucose or insulin responses between the different walking activity conditions, suggesting that 
intensity of activity during breaks per se did not influence metabolic outcomes, but rather the 
simple act of interrupting sitting with any activity might be beneficial for metabolic health.   
More recently, a separate research group sought to confirm these findings with a similar 
intervention strategy.  Pulsford et al. performed a randomized, cross over intervention of three 
arms in 25 inactive men (Figure 2).62  The three arms consisted of:  7 hours of continuous sitting, 
7 hours of sitting interrupted by 2 minutes of light intensity walking every 20 minutes, and 7 
hours of sitting interrupted by 2 minutes of 
standing every 20 minutes.  Similar to 
Dunstan et al., the subjects also received a 
standardized mixed test meal but they also 
received a fasted OGTT during the 
beginning of the day.  Serum glucose and 
insulin monitoring every 30 minutes 
throughout the day revealed significantly 
decreased glucose and insulin AUC during 
the walking-interruption arm.62  They 
found no significant improvement from the 
standing-interruption arm. 
 
 
Fig. 2 Pulsford et al.  J Sci Med Sport. 2016;16;30155-4 

14 
 
Pediatric Studies Interrupting Sedentary Behavior  
In youth, recent data (including our own) 
suggest that interrupting sedentary 
behavior has beneficial metabolic 
effects.64, 65  In protocol 13-CH-0169 we 
examined if an intervention to interrupt 
sedentary behavior in children would 
provide identifiable improvement in 
glucose metabolism (Figure 3).65  Using 
a randomized cross-over design in 28 
healthy weight children, we 
demonstrated that interrupting prolonged 
sedentary behavior in non-overweight 
children by intervening with 3 minutes of 
moderate intensity walking every 30 
minutes for 3hr significantly improved 
carbohydrate metabolism.65  In the 
children studied, interrupting sitting resulted in a 32% lower insulin iAUC (P =.001), 17% lower 
C-peptide iAUC (P = .001), and 7% lower glucose iAUC (P = .018) vs continuous sitting. Mixed 
model results indicated that insulin (P = .036) and free fatty acid concentrations (P = .009) were 
also significantly lower in the interrupted versus the continuous sitting condition.65  In addition 
to our published data obtained using a healthy weight cohort, we have obtained confirmatory 
results from a methodologically similar study carried out in a separate cohort of 37 children 
whom were overweight or obese.  In this cohort, interrupting sitting resulted in a 21% lower 
insulin iAUC (p<0.001) & 18% lower C-peptide iAUC (p=0.002). However, no significance 
difference was identified in glucose iAUC (p = 0.12).  
In addition to our study, Saunders et al. published a similar randomized cross over study 
in 2013.64  This research group applied a similar interrupted activity intervention only with light-
(rather than moderate-) intensity walking every 20 minutes for a full day (8 hours).64 The 
outcomes measured were also similar:  AUC for glucose and insulin.  But notably, in addition to 
0
3 0
6 0
9 0
1 2 0
1 5 0
1 8 0
0
2 0
4 0
6 0
8 0
1 0 0
T im e  (m in u te s )
In s u lin  (m c U /m L )
S IT
S IT  +  W A L K
S IT
S IT  +  W A L K
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
1 2 0 0 0
A U C  In s u lin  (m c U /m L )• m in
p  =  0 .0 0 1
0
3 0
6 0
9 0
1 2 0
1 5 0
1 8 0
0
2
4
6
8
T im e  (m in u te s )
C -P e p tid e  (n g /m L )
S IT
S IT  +  W A L K
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
A U C  C -P e p tid e  (n g /m L )• m in
p  <  0 .0 0 1
0
3 0
6 0
9 0
1 2 0
1 5 0
1 8 0
7 5
1 0 0
1 2 5
1 5 0
T im e  (m in u te s )
G lu c o s e  (m g /d L )
S IT  +  W A L K
S IT  +  W A L K
1 5 0 0 0
1 7 5 0 0
2 0 0 0 0
2 2 5 0 0
2 5 0 0 0
A U C  G lu c o s e  (m g /d L )• m in
p  <  0 .0 5
A
B
C
D
E
F
*
*
*
*
*
*
*
*
*
*
*
*
Fig. 3 Belcher et al.  JCEM. 2015; 100:3735–3743 

15 
 
the strength of the intervention being different (light intensity [30% of VO2 peak for Saunders et 
al] vs moderate intensity [80% of ventilatory threshold for us] walking), the serum sampling was 
much less frequent occurring only every 90 minutes instead of every 30 minutes as obtained by 
Dunstan et al. and during our study.  Finally, Saunders et al. only evaluated 19 total subjects.  
With these differences in methods, Saunders et al. failed to identify a difference in glucose 
metabolism by interrupted activity (i.e. no change in AUC of insulin or glucose).64  This directly 
contradicts all previous adult findings and our pediatric studies.48, 62, 65 As noted above, their null 
findings may be due to the small sample size and other methodological differences that limited 
their ability to detect the effects of interrupting sedentary time.   Regardless, all the positive data 
are reported from comparisons of outcome at single sessions of interrupted sedentary behavior 
and there are no available data testing if the changes observed acutely are sustainable. 
Continuous Glucose Monitoring to examine sustained effects on glucose homeostasis 
All the data presented above were obtained using indwelling catheters for venous 
sampling. As a result, only limited data from relatively infrequent sampling during in-lab studies 
are available.  Continuous glucose monitoring (CGM) using transcutaneous sensors that collect 
interstitial glucose concentrations is a method that can obtain 
frequent data sampling over prolonged periods.  The devices 
are approximately the size of a quarter and are inserted on the 
upper arm (Figure 4, Abbott Pharmaceuticals). Adult data 
indicate that altering physical activity, even minimally, in 
non-diabetic adults can significantly affect glycemic variation 
as identified on continuous glucose monitoring.66-68  Such 
data specifically include interrupting sedentary behavior, which has been demonstrated to 
decrease nocturnal glycemia as well as glycemic variability and post-prandial glucose.66, 67 
Blankenship et al. recently published novel CGM data collected on 10 sedentary 
overweight/obese non-diabetic adults who were given 3 different activity interventions while 
wearing CGMs.66  The arms of intervention included: A work day with no activity except one 30 
minute block of walking mid-day; A work day with ‘frequent long breaks’ which varied in 
length and frequency but totaled the same energy expenditure as one 30 minute block of 
walking; and A work day with ‘frequent short breaks’ which varied in length and frequency but 
Fig. 4 Continuous Glucose Monitor,  
Abbott Freestyle Libre 

16 
 
were more frequent and shorter in duration that ‘frequent long breaks’.  The participants were 
fitted with a CGM at the beginning of the day and wore the device for 24hrs.  During the testing 
day, they were given a standardized meal tolerance test in order to collect the main outcome data, 
AUC of glucose and insulin during the meal tolerance test.  (Participants were also given 
standardized fixed meals based on total daily energy expenditure for the remaining meals during 
each intervention 24hr-day).  The mixed meal tolerance test data indicated no difference in AUC 
of glucose or insulin.  However, there was lower glycemic variability in the ‘frequent long 
breaks’ compared to the day with one 30-minute block of walking with CGM data.   The mean 
amplitude of glycemic excursion (MAGE) was significantly lower when participants performed 
‘frequent long breaks’ for the day (p < 0.05).66  [Mean amplitude of glycemic excursion is the 
arithmetic average of all glycemic amplitudes greater than a pre-specified threshold (most 
commonly set at 1 SD)].69  Also, nocturnal duration of hyperglycemia (glucose > 7.8 mmol/l or 
>140 mg/dL) was significantly lower in both ‘frequent long breaks’ and ‘frequent short breaks’ 
compared to the day with one 30 minute block of walking (frequent long breaks = 2.5 ± 2.5 min;  
frequent short breaks = 45.6 ± 29.6 min; one 30 minute block of walking = 32.7 ± 16.4 min, p = 
0.05).66  It is important to note that this study did not include a nonintervention control arm and 
studied only 10 subjects; nevertheless, the data are highly suggestive the CGM may provide 
valuable data that cannot be obtained from traditional in-lab sampling during interrupted 
sedentary activity interventions.   
To our knowledge, there are no published data on CGM outcomes from interrupting 
sedentary behavior in youth.  Limited published data from CGM monitoring in non-diabetic 
youth have demonstrated associations between glycemic variability and vascular stress and show 
CGM devices are capable of demonstrating differences in glucose outcomes from dietary 
interventions.70, 71  For example, Desari et al. evaluated the relationship between CGM 
parameters including MAGE and markers of oxidative and vascular stress including c-reactive 
protein, cholesterol, triglycerides, non-esterified fatty acids, intercellular adhesion molecule-1 
and vascular cellular adhesion molecule-1, and E-selectin.71  In 34 children ages 13-21 years (12 
healthy weight, 10 healthy obese, 12 with Type 2 Diabetes Mellitus) with 5 consecutive days of 
CGM data, MAGE was significantly correlated with many markers of vascular stress including 
but not limited to oxidized low-density lipoprotein, E-selectin and intercellular adhesion 

17 
 
molecule 1.71 These data indicate that parameters of glycemic variability, as obtained by CGM, 
in youth can provide additional helpful information about carbohydrate metabolism.   
In addition to exploring glycemic variability and vascular health associations with CGM 
in non-diabetic youth, researchers have also begun to evaluate CGM outcomes for dietary 
interventions.  Bauer et al. evaluated the effects of a high protein breakfast in 28 overweight but 
non-diabetic teenagers.70 CGM outcome data were collected on day three of the intervention for 
a 24hr time period and included:  24hr AUC, morning glucose AUC, overnight glucose AUC, 
average, SD, maximal response, glucose fluctuation (max-min), and time spent above the 
glucose threshold of 100 mg/dl.70 No main effect by intervention was detected but post-hoc 
analyses revealed reduced 24hr-glucose variability (SD) in the individuals receiving a high 
protein diet.70   
The extant data have not evaluated CGM outcomes in sedentary behavior but provide 
insight and guidance into useful data that become accessible through CGM monitoring in youth. 
These novel exploratory studies demonstrate successful use of CGM in non-diabetic youth. 
When such data are combined with the positive CGM outcomes in adult interrupted sedentary 
behavior interventions, CGM monitoring of sedentary-interruption interventions in children 
could add important additional information for the effects of this intervention strategy in young 
cohorts.   
In conclusion, given the significant improvements in glucose homeostasis demonstrated 
in our acute intervention studies, along with the dearth of pediatric studies involving either 
continuous sampling or sustained interventions to interrupt sedentary behavior, a study that 
examines how beneficial repeated days of interrupted, versus non-interrupted, sedentary behavior 
might be to alter children’s glucose homeostasis measured with CGM is the logical next step. 
Such a study would be the first of its kind to assess sustained effects of brief sedentary 
interruptions in the clinical lab among youth and will provide novel data for within- and 
between- day effects of the intervention with continuous glucose monitoring.  However, it is 
important to note that metabolism is the not the only outcome that may be affected by 
interrupting sedentary behavior in youth.  Understanding that such an intervention strategy 
would be ultimately applied in a school or community setting on a routine basis, it is important to 

18 
 
evaluate effects on cognitive function, mood, anxiety, dietary intake, and non-intervention 
physical activity habits.   
Executive function and attention – definitions and alterations associated with obesity 
Executive functions are those mental processes that are central to goal planning, decision-
making, and choice of behavior.72 This construct is comprised of several underlying elemental 
processes such as inhibition, working memory, and cognitive flexibility (e.g.: task switching).73 
Development of executive functions is associated with the prefrontal cortex and emotional 
systems in the brain.74 Some data exist that indicate adult-level simple executive functions such 
as motor inhibition can develop as early as age 10, with gains in functional efficiency extending 
through early adulthood.75-77 Cognitive flexibility, goal setting, and information processing 
develop most rapidly from ages 7 to 9.78 Thus, executive functions are rapidly evolving during 
childhood and are necessary for behavioral development, emotional control, and social 
interactions.79 
Attention refers to the cognitive process of selectively concentrating on one 
environmental stimulus while ignoring other stimuli.80 There are several underlying processes 
such as sustained attention, selective attention, and divided attention81 that serve as the 
foundation for other mental processes. There are rapid changes in attention between the ages of 8 
and 10.82 In a sample of 400 3-12 year old Finnish children, maximum improvement in selective 
and sustained attention were reached by age 10.81 Thus, intervening in this age group may be an 
effective way to support the development of several sub-processes of attention. It is important to 
support attentional development because it is necessary for academic achievement in children. A 
longitudinal study in 2595 children reported that focused attention scores in preschool predicted 
achievement outcomes in childhood, whereas impulsivity scores in preschool predicted 
behavioral outcomes in childhood.83 
Relationship to obesity 
Executive function and attention are lower in overweight/obese children compared to 
their lean counterparts.79, 84 Data from NHANES indicate that higher BMI is associated with 
decreased cognitive function in children.85 Other studies also support these findings in 
children.86-88 Obese children have lower inhibitory control scores than their normal weight 
counterparts.89 Another study found that overweight boys had lower focused attention scores and 

19 
 
more inattention symptoms than their normal weight counterparts.90 Executive function deficits 
are also related to low physical activity and high sedentary behavior levels. In a latent class 
analysis of fourth to sixth graders, children in the “high sedentary, high fat/high sugar snacks, not 
weight conscious” category had more executive function problems than children in the “active, 
healthy eating” category.91 Executive function and attention in early childhood track into 
adolescence,79 suggesting that deficits may promote obesogenic behaviors that have long-term 
consequences. 
Obesity risk may be mitigated by the positive relationship between physical activity and 
executive function and attention in children. A seminal meta-analysis reported a positive 
association between physical activity level and aspects of executive function in children and 
adolescents, with a stronger effect in younger age groups (4-13 years). 92, 93 This positive 
relationship was independent of type of activity. Fourth grade children who were physically 
active had higher concentration scores,94 suggesting that attention performance is enhanced after 
physical activity. Physical activity is of much interest because it has effects on brain activation 
and development, and children may particularly benefit because their central nervous system is 
developing.95 A review of the existing literature on physical activity, fitness, and aspects of 
cognitive function indicated that physically fit children have greater mobilization of brain 
resources that allow them to perform cognitive tasks more rapidly than unfit children.96 Thus, 
research has focused on the effects of acute and chronic physical activity on executive function 
tasks in children.  
Executive function, attention, and physical activity 
Mechanisms 
There are two primary mechanisms that have been used to describe the relationship 
between physical activity and executive function and attention. The first mechanism relates to 
the physiologic changes produced by physical activity. Physical activity promotes central 
nervous system development, and the refinement of the prefrontal cortex97 through the 
attentional network of the brain that is active in cognitively challenging tasks.98 Thus, physical 
movement results in changes in brain activation and function99 that support the development of 
executive functions and attention during childhood. The second mechanism relates to the 

20 
 
learning and developmental changes produced by physical activity. Motor and pro-social skills 
that children learn through physical movement transfer to tasks involving aspects of executive 
function and attention, such as behavior inhibition and working memory, that relate to academic 
achievement.100 
Acute Physical Activity 
Executive function and attention are improved by acute physical activity.101 Researchers 
hypothesize that short bouts of exercise facilitate transient modulation of neural networks 
involved with specific cognitive tasks following the exercise.102 A review found that there was a 
small to moderate improvement in attention following acute physical activity in a classroom 
setting.103 In an early study, Gabbard and Barton found that mathematical scores increased in 
second grade children following 50-minutes of PE class participation.104 A more recent crossover 
study with 20 children (mean age= 9.5 years) reported increased response accuracy and academic 
achievement test scores following a 20-minute moderate-intensity walking bout.105 These studies 
support the view that acute bouts of exercise improve specific aspects of executive function and 
attention, and may be an intervention target to increase both physical and cognitive health in 
children. 
Chronic Physical Activity 
Chronic physical activity is positively associated with executive function and attention in 
children.106, 107 Longitudinal repetition of physical activity results in durable structural changes in 
the brain.102 A recent randomized control trial used functional MRI to measure brain activity 
while cognitive tasks were performed by overweight children randomized to either a 13-week 
exercise or control program. Results demonstrated a dose-response relationship existed: 
executive function and math achievement were increased in both the low exercise and high 
exercise groups, but more so in the high exercise group.108 A review of the longitudinal literature 
found that participation in school PE was positively associated with academic performance.109 
Donnelly et al. reported that a 3-year clustered randomized control trial on a classroom-based 
physical activity curriculum that promoted 90 minutes/week of MVPA resulted in a 6% 
improvement in standardized academic achievement scores and a slowing of BMI increases in 

21 
 
the intervention schools.107 These studies support the role of chronic physical activity in 
executive function and academic performance in children. 
Sedentary Behavior 
Research on the effects of sedentary behavior on executive function and attention is still 
developing. Longitudinal studies in children have found that more time spent watching TV is 
associated with greater attention difficulties and worse cognitive function.110 Results from a 
randomized control trial indicate that reducing sedentary behavior through 20- or 40- minute 
bouts of vigorous exercise increased academic performance in 222 sedentary overweight 7-11 
year old children.79, 111 A study conducted in 60 children (age 4) found that executive function 
was decreased immediately following a 9-minute television show.112 Because sedentary behavior 
has traditionally been operationalized as TV viewing in cognitive studies, it is unclear whether 
other sedentary behaviors influence components of executive function in children.  
Psychosocial health  
Acute and chronic physical activity produces beneficial effects on mood. Higher levels of 
physical activity have been associated with lower levels of depression,113 less perceived 
anxiety,114, 115 and better mood,116, 117 in children. In a sample of 64 children (ages 9-10 years), 
15 minutes of exercise increased feelings of positive mood whereas a 15-minute video decreased 
feelings of positive mood.118 The research clearly demonstrates that acute physical activity 
increases positive affect and mood in children. 
While there is an extensive literature examining the effect of physical activity on mood 
and affect, more research is needed to examine the effect of sedentary behavior on psychosocial 
outcomes in children.119  High levels of sedentary behavior are associated with increased 
depressive symptoms.120, 121 In a sample of 4320 children, sedentary behavior was positively 
associated with perceived anxiety and emotional symptoms.122 A recent crossover trial found that 
after an acute stressor, children spent more time being sedentary (watching TV).123 Early 
evidence suggests that sedentary behavior may increase negative mood and depressive 
symptoms. No previous studies have investigated if interrupting sedentary behavior in a 
sustained fashion affects psychosocial outcomes in children. 

22 
 
Dietary intake and activity 
 
Dietary intake is related to metabolic risk in children. In overweight children, higher 
soluble dietary fiber intake was associated with lower waist circumference124 whereas high sugar 
intake was associated with poor beta cell function, an early sign of increased risk of type 2 
diabetes.58 Findings from NHANES indicated that youth with higher fruit intake and Healthy 
Eating Index scores have a lower prevalence of metabolic syndrome.125 The findings from these 
studies suggest that balanced dietary macronutrient intake is essential for normal metabolic 
functioning. 
Dietary intake is associated with physical activity in children. Increases in physical 
activity in are associated with increases in total energy intake.126, 127 However, few studies have 
addressed the direct influence of specific dietary macronutrients on physical activity. In a sample 
of 210 girls, accelerometer-measured moderate-to-vigorous physical activity was negatively 
associated with percentage of calories from fat and carbohydrate.128 Other studies have found 
that diets high in simple carbohydrates have been associated with feelings of fatigue, and low 
levels of physical activity.129, 130 
Dietary intake is hypothesized to co-occur with some sedentary behaviors. A recent 
review found that high amounts of TV viewing was associated with lower fruit and vegetable 
consumption, higher consumption of energy-dense snack foods, fast food, and drinks.131 In a 
sample of 2908 adolescents (ages 14-19 years), screen-based behavior was inversely correlated 
with fruit and vegetable intake.132 A cross-sectional study in 1860 children (ages 5-12 years), fast 
food and snack consumption was positively associated with sedentary behavior.133 The limited 
evidence thus far suggests that higher levels of sedentary behavior are associated with unhealthy 
dietary intake.  There are no studies examining the effects of experimentally interrupting 
sedentary behaviors in a sustained fashion on dietary intake. 
Subject Selection 
 
Epidemiologic data indicate that sedentary behavior is associated with cardiovascular 
disease and all-cause mortality irrespective of adiposity.  For example, Thorp et al. report meta-
analysis findings on increased all-cause mortality, cancer, and cardiovascular disease in adults 
after controlling for BMI.17  Additionally, the effectiveness of this intervention in the preliminary 

23 
 
data described above was not dependent on adiposity.48, 64, 65  Also, the non-metabolic parameters 
such as cognitive function are independent of weight.  Therefore, our proposed intervention will 
likely affect healthy weight and overweight children equally within these outcomes.  Finally, our 
intervention is designed to be a preventative strategy intended to prevent dysfunctional 
metabolism before the onset of pathology such as Impaired Glucose Tolerance or Diabetes 
Mellitus Type 2 and be feasible for broad spread application into the community setting where 
the majority of subjects are expected to be healthy. For broad spread application, it is pivotal that 
preliminary data demonstrate potential benefits and negligible adverse outcomes in all subject 
populations within which it will be applied.  Given this, we will study healthy weight and 
overweight children who do not have evidence of metabolic disease. 
Magnetic Resonance Spectroscopy Arm: Use in Exercise Physiology 
A novel technique to explore the underlying physiologic mechanisms of interrupted activity is 
through skeletal muscle magnetic resonance spectroscopy (MRS).  MRS provides a technique to 
evaluate cellular level fat (specifically intramyocellular lipid, IMCL) content which in turn 
provides information on FFA storage and usage.134, 135  IMCL is positively correlated with 
insulin resistance and thus associated with the diabetes mellitus type 2.136, 137 Although details of 
the association (causal or not) have yet to be elucidated, it is readily hypothesized that acutely 
reducing IMCL improves insulin sensitivity.  However, it is also known that repeated, exercise 
training increases basal or fasting IMCL content in parallel with improved insulin sensitivity.138, 
139  Thus, the generally accepted understanding of IMCL and physical activity is that static, 
accumulation of IMCL is associated with poor insulin sensitivity but repeated depletion and 
replenishment of IMCL stores, which leads to increased basal IMCL is associated with improved 
insulin sensitivity.140  My collaborator for this proposal, Ahmed Gharib, MD, PhD, Head of  
Biomedical and Metabolic Imaging Branch within the National Institute of Diabetes and 
Digestive and Kidney Disease, has used MRS to evaluate skeletal muscle lipid content as a 
function of insulin resistance.141  MRS has also been successfully used to identify acute change 
in intramyocellular fat content surrounding physical activity142, 143 but has yet to be utilized for 
evaluating the effects of interrupting sedentary behavior.  As such, we propose, as an exploratory 
analysis, the use of MRS to explore associations of IMCL in children and fat mass and determine 

24 
 
if interrupted sedentary behavior in children acutely alters IMCL content. MRS will be used in 
accordance with its labeling and not operated in research mode. 
 
Conclusion 
In final summary, there are few data for the effects of experimentally interrupting sedentary 
behaviors on metabolism, cognition, mood, or dietary intake, and no such data for programs that 
study such interruptions in a sustained fashion. However, what are available suggest that one-
session interventions with at least moderate intensity bouts applied 2 or 3 times per hour may be 
successful at improving glucose homeostasis by decreasing circulating insulin. Therefore, we 
plan to conduct a pilot study that examines sustained metabolic outcomes from interrupting daily 
sedentary behavior in children applied with 3-minute interrupting bouts every 30 minutes for a 3-
hour duration over 6 days. With this study we will also collect exploratory mechanistic data from 
liver and muscle spectroscopy. Our chosen intervention mirrors our one-session preliminary 
trials and is in line with similar published intervention trials.64, 65   We have chosen the details of 
this strategy to maintain the potential of translation to the real-world setting.  Although some 
preliminary data have been obtained with an intervention applied every 20 minutes64 instead of 
every 30 minutes, this intervention is too frequent to be practical in a school setting. 
Additionally, the duration of 6 days has been chosen as an ideal intermediary to demonstrate 
sustained effects prior to broad spread application that will require extensive resources and as of 
yet are not strongly data-supported.  Finally, in addition to glucose homeostasis, we will explore 
the potential impacts of interrupting prolonged sitting on children’s executive function, mood, 
anxiety, dietary intake, and usual physical activity habits which aim to strengthen the rationale 
for broad application of this approach in the school setting. 
2. Study Objectives  
 
Project Goal:  The overall goal of this research is to examine whether providing brief 
interruptions in prolonged sitting produces long-term salutary changes in metabolism, without 
adversely impacting executive function, attention, anxiety, mood, or dietary intake. As a bridge 
between the acute studies that have demonstrated efficacy for this approach and long-term 

25 
 
school- or home- based translational programs, we will examine if brief interruptions of 
sedentary behavior applied for 6 consecutive days will produce sustained improvements in 
metabolism and to explore any changes in cognitive function or energy intake in children with 
intervention.   
Objective 1: To investigate if daily interruptions of sedentary behavior (sitting) versus prolonged 
sitting on 6 consecutive days improve glucose homeostasis.  Hypothesis 1a:   Compared to non-
interrupted sedentary behavior, interrupted behavior will result in significantly lower insulin and 
glucose incremental area under the curve (iAUC) during a 3-hour oral glucose tolerance test.  
Hypothesis 1b: As measured by continuous glucose monitoring, children with daily interrupted 
sitting will have lower average 24-hr glucose iAUC and lower mean amplitude glycemic 
excursion (MAGE) than children who engage in non-interrupted sedentary behavior. 
Objective 2:  To investigate if daily interruption of sedentary behavior adversely influences 
cognitive and psychological measures, dietary intake, or usual physical activity.  Hypothesis 2:  
Executive function, attention, mood, anxiety, dietary intake, and physical activity (at times 
outside the testing period) will not differ significantly between children randomized to daily 
interrupted versus daily non-interrupted sedentary behavior.  
Objective 3:  To investigate if acute or daily interruption of sedentary behavior affects 
intramyocellular lipid content within the vastus lateralis, tibialis anterior or soleus muscles.  
Hypothesis 3a:  Acute interrupted sedentary behavior will decrease intramyocellular lipid 
content.  Hypothesis 3b:  Daily interrupted sedentary behavior will increase resting/basal 
intramyocellular lipid content.   
 3. Subjects 
a. Description of study populations  
 
The proposed intervention has demonstrated acute efficacy in the general pediatric 
population, among both healthy weight65 and overweight children, ages 7-12 years.  
Therefore, we will enroll generally healthy child volunteers in a 1:1 ratio of healthy and 
overweight.  We will adhere to a maximum accrual ceiling of 150 participants.  Based on 
previous experience, we anticipate that no more than 20% of screened children will be 

26 
 
ineligible for the study. Thus, we will randomize up to 120 participants and, anticipating 
that at most 36 children will drop-out of the study before completion or have technical 
issues limiting data analysis, we expect at least 84 participants to complete the protocol 
(our target completion sample size is based on power analyses described below).  
Withdrawals/dropouts will not be replaced. 
i. Inclusion criteria 
Participants will qualify for the study if they meet the following criteria: 
(a) Good general health. 
(b) Age ≥ 7 and <12 years. 
(c) Fasting plasma glucose < 100 mg/dL 
(d) Body mass index (BMI) ≥ the 5th percentile, as determined by the CDC age- and 
sex- specific growth charts.144 
ii. Exclusion criteria 
 
Participants will be excluded from the study for: 
 
(a) Significant cardiac or pulmonary disease likely to or resulting in hypoxia or 
decreased perfusion. 
(b) Evidence of impaired glucose tolerance or type 2 diabetes, including fasting 
plasma glucose ≥ 100 mg/dL. 
(c) Presence of other endocrinologic disorders leading to obesity (e.g.: Cushing 
Syndrome). 
(d) Participants who have, or whose parent/guardians have, current substance abuse 
or a psychiatric disorder or other condition that, in the opinion of the 
investigators, would impede competence, compliance, or prevent the completion 
of the study. 
(e) Participants who have, or are currently receiving, anti-psychotic drugs that would 
affect metabolism, cognitive outcomes, and body habitus. 
(f) Participants receiving medical treatment other than diet for hypertension or 
dyslipidemia. 
(g) Participants with precocious puberty and/or receiving androgen and estrogen 
therapy. 

27 
 
(h) Participants currently taking medications for ADHD, or any disorder or use of 
medications known to affect body composition or weight including but not 
limited to glucocorticoids or other steroid compounds. 
(i) Presence of pre-existing neurocognitive disabilities, or an age-adjusted score 
below 85 on the Picture Vocabulary Test at the screening visit. 
(j) Presence of food allergies, such as peanut/tree nut, dairy, soy or any other food 
allergy or personal dietary restrictions that would preclude participant from 
consuming the daily diet or the buffet. 
(k) Presence of significant skin disease or allergy to adhesive material prohibiting 
placement of a continuous glucose monitor. 
(l)  Participants unable or unwilling to abstain from acetaminophen, ascorbic acid, or 
salicylic acid during study duration. 
(m)Participant does not speak fluent English. 
(o) Participant is or becomes pregnant. 
(p) Participant has an ambulatory impairment 
 
b. Optional MRS Supplemental Arm  
iii. 
Inclusion criteria 
(a) Participants will qualify for the study if they qualify for the primary Break It Up! 
study. 
iv. 
Exclusion criteria 
(a) Cannot have MRI scanning. Some of the reasons a child might not be able to have 
MRI include:  
• 
Implanted cardiac pacemaker or defibrillator  
• 
Cochlear Implants  
• 
Ocular foreign body (e.g. metal shavings)  
• 
Embedded shrapnel fragments  
• 
Central nervous system aneurysm clips  
• 
Implanted neural stimulator  
• 
Medical infusion pumps  
• 
Any implanted device that is incompatible with MRI.  

28 
 
(b) Is not likely to tolerate an MRI scan. Examples of medical conditions that would 
make it difficult to undergo MRI include severe anxiety (nervousness) or 
hyperactivity which make it hard for your child to lay flat for the study.  
(c) Requires sedation for MRI studies.  
(d) Has a condition that makes entry into the scanner difficult (e.g. weight over 550 
lbs, claustrophobia, etc.).  
(e) Has severe back-pain or motion disorders that make it hard for a child to lie on 
his/her back within the MRI scanner and hold still for the scan. 
4. Study Design and Methods 
 
a. Study Overview:   
i. Trial Design: We will carry out a parallel group randomized controlled trial that 
compares child carbohydrate metabolism and cognitive function during 5 days of 
sedentary after-school activities versus after-school activities with structured 
interruptions provided by walking for 3 minutes on a treadmill every 30 minutes at a 
moderate activity rate selected according to each participants’ level of fitness. A final 
6th day will compare results between groups from an oral glucose tolerance test 
conducted in the fasted state. Subjects will be randomized to one of two conditions: 
(a) Study Intervention: Interrupted sedentary behavior. This test condition will 
consist of 6 consecutive daily after-school /afternoon visits for a three-hour 
experimental session.  The first five sessions will be performed mid-afternoon 
(Monday – Friday, after school or late afternoon); the 6th will be in the morning 
(Saturday, approximately 8am). Each session will consist of prompted 3-minute 
moderate-intensity walking bouts performed on a treadmill every 30 minutes for 3 
hours. There will be a total of 6 walking bouts (18 minutes total) each day.  
‘Moderate-intensity walking’ speed and grade will be selected to achieve 80% of 
the heart rate achieved at the ventilatory threshold as determined during a V02max 
test.   

29 
 
(b) Study Control: Non-Interrupted sedentary behavior.  This control condition 
will consist of children attending similarly-timed visits for 6 consecutive days 
providing 3 hours of sedentary behavior/sitting during which the children will not 
engage in any physical activity.  Rather, they will engage in continuous sedentary 
behaviors (e.g. watching TV or working on homework while seated) for 3 hours 
each day without interruption.   
ii. Visit Work Flow (Also see Figure 1):  There are no inpatient visits. Participants will 
be evaluated at a series of day-hospital visits (7 in total) using both the 1NW pediatric 
day hospital and the 5SWN metabolic units of the Hatfield Clinical Research Center.  
The initial screening evaluation visit will use the 1NW pediatric day hospital and will 
involve approximately 6 hours of testing.  Following screening, the daily test visits 
will be conducted in the 5SWN Metabolic Unit and involve approximately 3.5 hours 
each day for the 5 daily visits.  The final visit will also take place in the metabolic 
unit and involve approximately 5 hours of testing.  In summary, each participant will 
complete a screening visit and, if randomized, will complete 6 consecutive days of 
study visits.  The block of daily visits will be initiated no further than 90 days from 
the date of screening; therefore, each participant should complete study participation 
within 97 days of screening.   
iii. Relationship with other protocols:  This is an independent protocol with no direct 
relationship to other study protocols.  
b. Recruitment 
i. Description of recruitment strategy. Our team has considerable experience in 
recruiting and retaining child participants in research studies at the Clinical Research 
Center. Most germane to this study, Drs. Yanovski and Broadney successfully 
recruited 95 children ages 7-11.99y for the pilot studies of protocol 13-CH-0169 over 
3 years to evaluate this sedentary intervention strategy in the acute setting.  Dr. 
Broadney has been in close communication with the NIH Clinical Center Patient 
Recruitment and Public Liaison (PRPL) Office to develop recruitment materials and 
strategies to successfully reach the target population. All recruitment efforts will 
include an email and telephone number for parents to call if interested (See 

30 
 
Appendices A for all media, email, and letters documents drafted for PRPL use). 
These include flyers, social media content, and mailings to local pediatricians, school 
listservs and community centers throughout the greater District of Columbia, 
Maryland, and Virginia metropolis.  
ii. Recruitment Methods: Participants will be recruited via multiple methods:  
(a) Letters will be mailed to the parents of appropriate-aged children in local areas 
including Montgomery County, Fairfax County, and Washington, DC. 
(b) Flyers posted at NIH and other local facilities (e.g. elementary schools, 
community centers, churches, public libraries) with permission.  
(c) Local community groups will be asked to send the flyer to their listservs.  
(d) Schools, churches, pediatricians, and community centers will be contacted 
regarding the study and fliers will be distributed. 
(e) Potential volunteers that have registered on the NIH Clinical Center 
ResearchMatch program will be contacted. 
iii. Anticipated Accrual Rate:  We expect approximately 5 interested participants will 
be screened per month, with increases in accrual during school vacation dates and 
declines when the surrounding public school districts are in session.  
iv. Initial contact:  Potential participants who have sent their information to the study 
team will be contacted via telephone and/or email communication upon first inquiry 
(See Appendix A for telephone script, email template and FAQ’s that will be 
provided).  If basic participation criteria are met (i.e. child within age range and 
without significant medical diagnoses), parents/guardian(s) and participants will 
receive consent and assent forms (written explanations of the purposes, procedures, 
and potential hazards of the study) before the screening appointment. If a family 
wishes to join the study, the participant and a care provider able to give consent for 
research participation will be invited to the Clinical Research Center for formal 
screening. 
c. Screening 
 
Participants will complete the following during their screening visit.  
i. 
Vital signs, height, weight. Blood pressure, height (in cm), weight (in kg), 
temperature, respiratory rate, and pulse rate will be obtained by NIH nursing staff. 

31 
 
BMI will be computed to determine eligibility. Subjects with BMI <5th percentile for 
age and sex will be excused from the study. 
ii. 
Protocol review and signing of consent and assent forms. Parents/guardian(s) and 
participants will review the consent and assent forms with study personnel designated 
to do so by the PI. All study procedures will be discussed, and study risks and 
benefits explained. Communication of this information and of the participant’s 
consent will be documented in the medical record before any study procedures, 
including other screening procedures, are done. All participants will be informed of 
their right to withdraw from the study at any point. Participants will be informed of 
compensation. Parent/guardian(s) and participants will be given signed copies of the 
consent and assent form for their records. 
iii. 
Complete medical history and physical exam will be conducted by a trained 
practitioner. The exam will include Tanner pubertal staging. Waist and hip 
circumference (cm) will be measured in triplicate by nutrition staff (see below).  
iv. 
Practice measures. Participants will be oriented to all procedures that will occur 
throughout the screening visit day (with the assistance of Child Life services) to 
reduce any anxiety.  
v. 
Fasting blood draw. A fasting blood sample will be drawn to screen for exclusionary 
conditions and determine metabolic, hematologic, renal, and hepatic function 
including: acute care panel, insulin, c-peptide, free fatty acids, triglycerides, 
hemoglobin, creatinine, thyroid function tests (including anti-thyroid antibody panel), 
mineral, and hepatic panels, thyroid function tests (TSH and Free Thyroxine), C-
reactive protein, Hemoglobin A1C, complete blood count, serum beta-human 
chorionic gonadotropin or urine pregnancy test, and serum and plasma samples to be 
saved for future research. ELA Max cream will be applied to numb the blood draw 
site.  
vi. 
Socioeconomic status (SES). A demographics questionnaire that assesses 
race/ethnicity and parent/guardian education level and income will be completed by 
the parent/guardian so that the Hollingshead two-factor index of social position can 
be calculated.145  See Appendix G. 

32 
 
vii. 
Receptive vocabulary. Participants will be given a Picture Vocabulary Test to 
measure baseline receptive vocabulary, which may serve as a proxy for IQ. The 
participants are presented with an audio recording of a word and four photographic 
images on the computer screen and asked to select the picture that most closely 
matches the meaning of the word. This test was selected from the NIH Toolbox 
(http://www.nihtoolbox.org). Participants whose age-adjusted score is below 85 
(below average function) will be excluded from the study.  
viii. 
Heart rate monitor. Participants will be familiarized with the heart rate monitor 
(Polar Vintage NV, Polar, Port Washington, NY, USA), and will practice wearing the 
same monitor that will be worn during the test visit(s). 
ix. 
Cognitive Testing.  Participants will be shown a 2-minute overview video of the 
Cognitive Brief Battery (Cogstate, New Haven, CT that will be administered to them 
on Test Day #1 and Test Day #5 (https://cogstate.com/featured-batteries/). 
CogState Brief Battery (CBB) is an approximately 20-minute computerized battery 
for children and adults ages 4 - 90 years, which includes tasks tapping processing 
speed, attention and vigilance, learning and memory, working memory and executive 
functions.146 This measure was designed for repeated assessments over a short time 
period with virtually no practice effects in adults and children due to multiple 
forms.147, 148 Age-based standard scores (mean = 100, SD 10) are computed for each 
task based on a normative sample of several hundred individuals.  CogState has 
strong construct validity, minimal practice effects, and good stability.  It was 
designed to be language independent in order to ensure the validity of the tests in a 
cross-cultural setting.149-152 Further, CogState has been used in numerous pediatric, as 
well as adult, oncology clinical trials.   
a. 
Detection Task: This subtest of the CogState takes 2 minutes to administer 
and measures psychomotor function and processing speed.  It uses a well-
validated simple reaction time paradigm using playing card stimuli.  In this 
task, the playing cards are all red and black jokers.  The subject is asked to 
press the Yes key as soon as the card in the center of the screen flips over. 
b. 
Identification Task: This task takes 2 minutes to administer.  It is a measure 
of visual attention and uses a well-validated choice reaction time paradigm 

33 
 
using playing card stimuli.  In this task, the playing cards are all either red or 
black jokers.  The subject is asked whether the card currently being presented 
in the center of the screen is red.  The subject responds by pressing the Yes key 
when the joker card is red and No when it is black. 
c. 
One Back Task: The One Back Memory task takes approximately 2 minutes 
to administer and is a measure of working memory.  It uses a well-validated n-
back paradigm using playing card stimuli.  In this task, the playing cards are 
identical to those found in a deck of playing cards except for the joker.  The 
subject is asked whether the card currently being presented is the same as the 
one presented immediately previously.  The subject responds by pressing the 
Yes or No key. 
d. 
One Card Learning Task: This task takes 5 minutes to administer. The One 
Card Learning task is a measure of visual recognition memory and uses a well-
validated pattern separation paradigm using playing card stimuli to measure 
hippocampal functioning.  In this task, the playing cards are identical to those 
found in a deck of playing cards except for the joker.  The subject is asked 
whether the card currently being presented in the center of the screen was seen 
previously in this task.  The subject responds by pressing the Yes or No key. 
x. 
Mood. Participants will complete a practice Positive and Negative Affect Scale-Child 
(PANAS-C). The test will be explained and any words that the child does not 
understand will be explained. The 20-item scale consists of a 10-item Positive Affect 
(PA) scale and a 10-item Negative Affect (NA) and takes approximately 10 minutes 
to complete.153  Participants indicate how often they have experienced several states 
on a 5-point Likert scale (1= very slightly/not at all; 5= extremely). This 
questionnaire has been validated in children,153, 154 and demonstrates alphas of .91 and 
.88 for PA and NA, respectively. Item-total correlations range from .49-.78 on PA 
and .36-.80 on the NA scales, respectively. The test-retest reliability is high 
(correlations of .66-.82).  Subjects will complete the PANAS-C and the “Feelings 
Questionnaire” through the NIH Clinical Trials Survey System.   
xi. 
Anxiety. Participants will complete a practice State Trait Anxiety Inventory for 
Children (STAIC).155 The test will be explained and any words that the child does not 

34 
 
understand will be explained. The scale measures acute (state) anxiety and longer-
term (trait) anxiety. It asks the child to rate 20 statements from ‘hardly every true’ to 
‘often true’ and takes approximately 15 minutes to complete. The scale has been 
validated in 1551 children ages 6-14 years. Subjects will complete the STAIC 
through the NIH Clinical Trials Survey System.   
xii. 
Physical Activity Evaluation.  To determine the average amount of moderate-to-
vigorous physical activity each participant has engaged in within the previous week, 
children will be asked two items from the International Physical Activity 
Questionnaire (IPAQ) screening form (See Appendix G).156  This information will be 
used to:  
a. 
Determine caloric requirements for standardized meal calculation  
b. 
Stratify participants for randomization purposes.  
xiii. 
Dietary Evaluation & Metabolic Diet Education.  Participants will be interviewed 
by and receive education instruction from the nutrition staff.  This will allow us to 
obtain each participant’s meal preferences and provide metabolic diet education (i.e. 
goals of the metabolic diet throughout the study period) to improve adherence. 
Additionally, waist and hip measurements will be obtained in triplicate. 
xiv. 
Electrocardiogram (EKG). All children will undergo an EKG. If the EKG or 
physical examination suggests possible concerns in cardiovascular function, 
echocardiography &/or formal cardiology consultation will be obtained before the 
participant performs the maximal exercise test.  
xv. 
Dual-energy x-ray absorptiometry (DXA) scan. As part of a whole-body 
composition and bone mineral density assessment, DXA scan will be performed to 
identify lean and fat mass. DXA involves passing a collimated X-ray beam of 40 and 
70 KeV through the subject and collecting the X-rays that pass through the patient 
with a standard X-ray detector. Because the delivery and attenuation of the X-rays are 
measured separately for the two principal energies, the procedure allows the 
differentiation of substances in their path if the attenuation coefficients of these 
substances are known. The manufacturer specifies that the array beam delivers an 
effective dose of 0.003mrem total body irradiation. Participants will undergo DXA 
scans in the Metabolic Clinical Research Unit (MCRU) using the Lunar iDXA (GE 

35 
 
Healthcare, Madison, WI, USA) machine. The scan takes approximately 20-30 
minutes. An external standard, simulating bone, fat, and muscle, is scanned once a 
week to validate measurements. This standard allows calculation of total body fat, 
muscle, and bone mass, and percentage of body fat. 
xvi. 
Treadmill fitness test to determine maximal oxygen uptake (V02max). 
Cardiorespiratory fitness will be measured via a modified Balke continuous ramp 
treadmill protocol with some individual tailoring if necessary. We will monitor the 
heart electrical rhythm strip and continuous vital signs as participants exercise, and 
stop exercise if there are any signs of cardiovascular compromise. A Pediatric 
Advanced Life Support (PALS) certified physician or nurse practitioner will be 
present during this test. Each participant  will exercise on the treadmill to the limit of 
their exercise tolerance, according to previously established criteria 157. Participants 
will be familiarized with the equipment and procedures prior to any testing. The 
metabolic cart will be calibrated prior to data collection. Resting heart rate and blood 
pressure will be determined when the participant has been seated in a chair for at least 
5 minutes before testing. EKG lead sites will be prepped and participants will be 
fitted with the leads, and a facemask, to measure expired gas exchange during 
exercise. Prior to testing, the participant will be given a 4-minute warm-up at a self-
selected pace with 0% grade to orient them to the equipment and prepare them for the 
first stage of testing. Treadmill speed and grade will then be continuously increased 
until the subject reaches the limit of their exercise tolerance. Treadmill speed and 
grade increase will be based on body height, weight and predicted peak VO2. 
Participants will be encouraged to exercise to their tolerance limit, but will be allowed 
to stop the test at any time. EKG, blood pressure, rating of perceived exertion (RPE), 
and any signs and symptoms of exercise intolerance will be monitored throughout 
exercise and during recovery. Expired gas exchange will be measured breath by 
breath by metabolic cart. Lactate threshold (LT estimated by gas exchange, will be 
determined using the V-slope method and dual criteria graphs158 Peak oxygen uptake 
(peakVO2) will be defined as the highest VO2 achieved at the end of exercise. 
Treadmill speed and grade equivalent to 80% LT will be used to program walking 
during the interrupted sitting portion of the study (see text below). Pediatric exercise 

36 
 
testing guidelines, published by the American Heart Association, will be observed 
regarding termination of exercise:157 
a. 
The child requests to stop the test. 
b. 
Ventricular tachycardia. 
c. 
Any significant drop in systolic blood pressure or a failure of systolic blood 
pressure to rise in the presence of increasing workload. 
d. 
Signs and symptoms indicating potential hazard to the patient such as pain, 
headache, dizziness, syncope, excessive dyspnea or fatigue. 
e. 
A > 3 mm ST segment depression or elevation. 
f. 
Significant arrhythmia precipitated by exercise such as PVC’s with increasing 
frequency, supraventricular tachycardia, ventricular tachycardia or 
atrioventricular block. 
g. 
Excessive rise in blood pressure: systolic blood pressure >250 mm Hg. 
h. 
Failure of the monitoring system. 
d. Study procedures 
All procedures are for research purposes; there is no provision of standard medical care. 
Participants found eligible after screening will be scheduled for 6 visits (Days #1-6) on 
consecutive days. Participants will be randomized to one of the two conditions for all of 
their subsequent visits. Randomization will be done by randomizing each subject to a 
single treatment by using the method of randomly permuted blocks with average block 
size 4. Subjects will be stratified by sex (M, F), age (7-9.49 vs. 9.5-11.99y) weight status 
(BMI<85th percentile vs. ≥85th percentile), and IPAQ result (low, moderate, and high 
usual physical activity) to promote similar subject characteristics in the two groups. 
See Appendix B study visit flow table. 
 
i. 
Test Days #1-6 Daily Procedures:  
a. 
Vital Signs. Blood pressure, height (in cm), weight (in kg), temperature, 
respiratory rate, and pulse rate will be obtained. 

37 
 
b. 
Questionnaires. Parents and/or participants will answer a brief health 
questionnaire about participants’ medication use, general health, and sleep 
duration in the previous 24-hours, see Appendix G.  Additionally, a 24hr food 
recall will be administered. 
c. 
Randomized condition.  Each participant will execute their assigned 
randomized condition as follows: 
(i) Sedentary condition.  The participant will execute 3 hours of sedentary 
(seated or recumbent) activity of their choice. Activity monitoring 
(described below) will be obtained. Continuous heart rate monitoring 
(described below) will be obtained on test visit #6.  
(ii) Interrupted condition:  Throughout the visit, the study team will prompt 
the participant to walk on the test treadmill for 3-minutes every 30 minutes 
for 3 hours. There will be a total of 6 walking bouts (total exercise time: 18 
minutes). This walking activity will be ‘moderate-intensity walking’ 
determined as 80% of maximum heart rate determine during the V02max 
test described in screening. We will calculate and set the treadmill speed and 
gradient per the estimated target heart rate prior to beginning the test 
protocol.  Heart rate will be monitored during each walking bout, and 
maximum heart rate will be manually recorded after each walking bout. 
Activity monitoring (described below) will be obtained. Continuous heart 
rate monitoring (described below) will be done on test visit #6.  Participants 
will be required to remain sedentary (seated or recumbent) in between their 
walking bouts. 
d. 
Continuous heart rate monitoring:  A heart rate monitor (Polar Vintage NV, 
Polar, Port Washington, NY, USA) will be used to determine activity state 
during walking bouts for participants randomized to the “interrupted” condition 
on all test visit days. Participants randomized to the “sedentary 
 condition will wear a heart rate monitor during test #6 only..  After vital signs 
are obtained, participants will wear the monitor for the duration of test visit(s) 
(as specified above),  and remove it prior to patient dismissal.  Device initiation 
and data collection will be performed weekly, such that one device will be 

38 
 
initiated prior to the test day(s)  of each participant’s study week, they will use 
the same device for the entirety of their visit(s) and data will be collected from 
the device at the conclusion of the visit(s)(no further than 5 business days post 
study conclusion).   
e. 
Continuous glucose monitoring:  Beginning on day #1 of testing/intervention, 
a continuous glucose monitor (Libre Pro, Abbott products) will be placed on 
each child to record glucose trends continuously throughout their 6-day study.  
The Libre Pro device will be evaluated for placement, skin irritation, function, 
and data collection daily at each testing visit.  It is expected that only 1 device 
will need to be placed to complete the entire 6-day study. On Day #6, the final 
day of testing, the Libre Pro device will be removed.  Continuous glucose 
monitoring data will allow calculation of average daily glucose iAUC as well as 
mean amplitude glycemic excursion (MAGE; the arithmetic mean of all 
increases or decreases in glucose that exceed 1 SD of the total set of glucose 
values).  If a device is lost/falls off between study sessions, a replacement 
device will be placed on the subject.  If a child consumes a listed medication 
known to interact with glucose readings (acetaminophen, vitamin C, and 
aspirin), the following 24hrs of CGM data will be admitted from their analyses 
but the device will otherwise remain on the subject to continue collecting data 
throughout the remainder of their study block.   
f. 
Continuous physical activity monitoring:   On day #1 of testing/intervention 
participants will be given a wrist accelerometer (Actigraph GT3X+) to wear 
continuously throughout their 6-day intervention to assess in-study as well as 
free-living activity, sedentary behavior, and sleep efficiency.  This device will 
also be removed on day #6 and data will be downloaded.  These data will be 
used to determine if physical activity outside of the test condition changes as a 
result of intervention. Additionally, the participant will be fitted with a hip 
accelerometer on test visit #6, which will be worn for the entirety of test visit 
#6. These monitors will provide added data to evaluate fidelity of the 
interventions.  

39 
 
g. 
Standardized meal intake:  Beginning on day #1 of intervention, the 
participants will be given daily standardized meals to consume throughout the 
duration of the intervention.  Specifically, they will be given a standardized 
snack (10% of daily caloric intake) upon arrival for their daily test visit (days 1-
5 of intervention) which will be consumed during their 3 hours of intervention.  
Then they will be sent home with 3 packed meals and one snack (dinner, mid-
morning snack, breakfast and lunch) each day to be consumed as their sole 
caloric intake until they return for their next daily clinic visit.  (Meals with 
consist of the following caloric proportions for daily total intake: breakfast 20%, 
lunch & dinner 30% each, snacks 20% of total daily requirement).  This cycle 
will continue until they return for their final fasting visit on day #6 (see below).  
Nutrition staff will verify intake of provided foods and collect any leftover 
foods to be weighed back. Participants will also be asked to report any 
additional food (not from the standardized meals) that was consumed. The 
energy content of the standardized meals and snacks will be calculated based on 
individual weight & caloric expenditure assessed from weekly physical activity 
ascertained from questionnaire data (International Physical Activity 
Questionnaire screening form).156  
Food handling details:  All foods are weighed on electronic balance scales in 
gram weights to the nearest gram; foods consumed are determined by subtracting 
the food weights after the participant’s meals from initial weights. Nutrition staff 
preparing and measuring food have yearly HACCP food safety training that 
focuses upon safe food handling, proper temperatures, hand washing, and using 
gloves for handling foods. All nutrition staff has additionally received training in 
weighing/processing foods for controlled feeding studies to ensure consistency in 
the method and presentation. 
 
ii. Test Day #1:  Cognitive function, mood and anxiety questionnaires. (See details 
under Screening visit).  On the first day of testing, participants will perform the same 
anxiety and mood questionnaires as performed during the Initial Screening and be 
administered the Cogstate Brief Battery (both practice and baseline) as follows:   

40 
 
a. 
*Cogstate Brief Battery practice battery.   Administered 10 minutes prior to 
beginning of 3-hour session. 
b. 
Cogstate Brief Battery.  Administered at time 0 and again at time 120 minutes 
of study visit. 
c. 
PANAS-C.   Administered at time 0 and again at time 180 minutes of study 
visit. 
d. 
STAIC.  Administered at time 0 and again at time 180 minutes of study visit.   
     * 
iii. Test Day #5: Cognitive function, mood and anxiety questionnaires.  (See details 
under Screening visit).  On the second to last day of testing, the participants will 
perform the same cognitive function, anxiety, & attention questionnaires as 
performed during Test Day#1 as follows:  
a. 
Cogstate Brief Battery.  Administered at time 0 and again at time 120 minutes 
of study visit.  
b. 
PANAS-C.   Administered at time 0 and again at time 180 minutes of study 
visit. 
c. 
STAIC.  Administered at time 0 and again at time 180 minutes of study visit.   
 
iv. Test Day#6:  Participants will be asked to fast overnight in preparation for this 
morning visit. In addition to the daily procedures described above for Test Days #1-6 
(including engaging in their randomized assignment to sitting-only or sitting 
interrupted with walking), the participant will perform the following final study 
procedures:  
a. 
Placement of IV catheter. ELA Max cream will be applied to numb the IV 
site. The NIH Vascular Access Device service will be consulted as needed to 
place the IV catheter for this test.  
i. Fasting oral glucose tolerance test (OGTT). A standardized 
dextrose oral solution (75 g per 300mL; dose 1.75g per kg of body 
weight up to a maximum dose of 75g) will be given at time 0. The 
participants will be asked to execute their randomized condition 
(seated or interrupted seated as described above). Blood will be 

41 
 
sampled at times -5, 0, +20, +30, +60, +90, +120, +150, and +180 
minutes for measurements of insulin, C-peptide, glucose, cortisol, 
FFA, and triglycerides. Research specimens (10ml) will be 
obtained for future analyses at time 0, 30 and 120 minutes. During 
the 180 minutes of prolonged sitting, participants will be permitted 
to use the restroom if necessary and drink water. After completion 
of this sampling, the intravenous line will be removed.  
b. Cognitive function, mood and anxiety questionnaires.  (See details under 
Screening visit).  On the last day of testing, the participants will perform the same 
cognitive function, anxiety, & attention questionnaires as performed during Test 
Day#1 & #5 as follows:  
i. Cogstate Brief Battery.  Administered at time 0 and again at time 
120 minutes of study visit.  
ii. PANAS-C.   Administered at time 0 and again at time 180 minutes 
of study visit. 
iii. STAIC.  Administered at time 0 and again at time 180 minutes of 
study visit.   
c. Hunger ratings. Hunger will be assessed using a visual analog scale (VAS), 
through Clinical Trials Survey System (CTSS). The VAS is a simple category 
scale of satiety with anchors at the end-points and at five equally spaced intervals 
between the anchors.159 The VAS assesses satiety using a 0mm to 100mm range, 
with “Not at all hungry” at 0mm and “Greatest imaginable hunger” at 100mm. 
The VAS will be administered at pre-test, +30, +60, +90, +120, +150, and +180 
minutes after the walking and blood draws. They will take approximately 5 
minutes to complete. VAS scale scores are reproducible across multiple time 
points and predict subsequent food intake in children.160-162 A comparison 
between different VAS scales found the bipolar hunger/fullness scale to have the 
best correlation (r= 0.64) and reliability.163 
d. Buffet test meal. A standardized multiple-item buffet test meal (approximately 
10,000 kcal) will be given at the end of each condition block, day #6 of testing. 
The meal has an array of macronutrients (approximately 12% protein, 51% 

42 
 
carbohydrate, and 37% fat) from foods frequently consumed by children.65 Food 
energy and macronutrient content are calculated using the USDA National 
Nutrient Database for Standard Reference. Participants are instructed to ‘eat as 
much as you want’ and the amounts of foods and beverages consumed are 
calculated by measuring the differences in weights (g) of each item before and 
after the meal. Duration of eating is measured from the time the experimenter 
leaves the room until the participant indicates eating is completed. Energy and 
macronutrient consumption will be calculated. 
Food handling details:  See Standardized Meal Intake above 
e. 
CGM device removal. 
f. 
Accelerometer device removal.  
 
Optional Supplemental MRS Arm Study Design: 
a. We will enroll up to 75 children from the Break It Up protocol for this 
supplemental arm. Participants qualifying for the Break It Up parent protocol will 
be offered the opportunity to participate.  A separate consent and assent form have 
been prepared and will be signed before these studies are done. 
b. Baseline MRS scan:  Participants will attend a visit 0-21 days prior to beginning 
their Break It Up! test week to perform a baseline 1H-MRS scan of liver and up to 
3 skeletal muscles (vastus lateralis, tibialis anterior and soleus muscles) using 3T 
Magnetic Resonance scanner and a multi-channel flexible 
receiver coils.  Magnetic Resonance Spectroscopy (MRS) will be performed to 
quantify liver fat and intra- and extramyocellular lipid content.  This pre-
intervention, baseline scan will be performed in a standard fashion after 
participants consume a standard meal shake (calculated to be approximate 30% of 
daily energy needs).  In effort to minimize external factors, subjects will be asked 
to refrain from strenuous activity, such as aerobic exercise 30minutes 
consecutively or more, on the day prior and the morning of this scan.  
Additionally, a 24hr dietary recall will be performed. 
c. Post Intervention MRS scans:  On day # 4 or #5 of the main study (based on 
equipment availability), participants will repeat the same MRS scan of their 

43 
 
skeletal muscle as their pre-intervention baseline scan two more times.  The first 
will be performed at approximately 12pm under the same conditions of the 
baseline scan (including consuming the same breakfast shake and refraining from 
strenuous activity, such as aerobic exercise 30minutes consecutively or more, on 
the day prior and the morning of this scan).  After this scan, the subject will be 
transferred to the metabolic unit (via wheelchair) for their main study intervention 
session.  The subject will then carry out their randomized intervention session and 
consume their standard diet (per the Break It Up! protocol).  The third and final 
MRS scan will be performed immediately after the subject has completed their 3-
hour intervention session at approximately 4pm. 
*Of note, on this day the original CGM device will be removed prior to the MRS scan 
and new device placed after the final MRS given that the CGM device is not 
compatible with MRI scanning. 
 
End of participation 
a. 
Participants will complete study participation at the end of the Test Day #6 visit.   
b. 
Participant families will be told they should return for routine medical care to 
their usual pediatrician. 
c. 
The guardians of each participant will receive the following information.  
Additionally, this information will be shared with a participant’s health care 
provider upon request:  
1) 
Abnormal fasting blood test results, such as high fasting blood sugar, from 
the outpatient screening visit. 
2) 
Abnormal findings from the physical exam or EKG at the outpatient 
screening visit. 
3) 
Questionnaire results from the outpatient screening visit if there is any 
evidence of significant emotional concerns on the anxiety or mood 
questionnaires. 
d. 
Formal medical records will be available to all participants through the NIH 
Medical Records Department:  

44 
 
1) 
Clinical center laboratory results 
2) 
Physical exam documentation 
3) 
Electrocardiogram  
5. Management of Data and Samples 
a. 
Storage 
i. Human biospecimens will be collected and stored using procedures appropriate for 
the type of biospecimen being collected and will be handled in accordance with the 
U.S. Occupational Safety and Health Administration’s Bloodborne Pathogens 
Standard for Samples. Sample freezers will be operated using facility environments 
that include ambient temperature controls, good air circulation, lighting, and 
security. Systems are in place to allow for local and remote temperature monitoring 
of freezers. The NIH has an emergency preparedness plan that covers equipment 
failures and power interruption, including back‐up storage capacity and back‐up 
power generators. 
ii. The principal investigator and the co-investigators will have access to identifiable 
participant records. We will follow guidelines developed by the NIH for projects 
funded by the Federal Government for managing such samples. 
iii. Samples and data will be coded. Data will be coded by subject number, but will be 
readily associated with identifiable participant records. Records containing personal 
identifiers will be maintained consistent with the security measures required by the 
NIH. Data will either be transferred directly from the electronic medical record into 
the NICHD Clinical Trials Data Base (CTDB), or for data where direct transfer is 
unavailable, will be double-entered by hand into CTDB.   
iv. All data will be kept on secure computers in the SGO office, whose doors will be 
locked when staff is not present.  All paper data will also be kept in locked areas in 
the SGO office.  
• Serum and plasma samples not analyzed immediately (as described above) will be 
stored in locked freezers by the NICHD sample management team supervised by 
the NICHD Clinical Director and tracked/managed through CTDB. Human 

45 
 
biospecimens in storage will have a unique identifier, will be labeled with a printed 
label and will contain a barcode. Samples will be tracked using a computer‐based 
inventory system that records the location and detailed information of every 
specimen in the repository. 
• Data will be stored in a password-protected computer system and in paper files in 
locked file cabinets and office areas to which only the NIH research team will have 
access.   
• Samples and data collected from participants in this research study will be saved 
and used for future biochemical testing related to body weight and metabolism. This 
testing may be done by research laboratories that are not at the NIH.  Additional 
consent will be obtained to use these samples if the consent form indicates that such 
consent is required.  Prior to transfer of samples and data to non-NIH collaborators, 
a material transfer agreement (MTA) with the outside institution will be established.  
We will provide the NICHD IRB with a listing of MTA’s that have been 
established under these provisions at the time of each Continuing Review. 
• If unauthorized disclosure of identifying information linked with samples or data 
occurs, the IRB will be notified by the Principal Investigator within 7 days of 
learning of the occurrence. 
• Samples and data will be stored for a minimum of 3 years after completion of 
analyses for this study, but may be kept indefinitely.  Furthermore, in compliance 
with federal law, records will be maintained until all subjects enrolled in the study 
have reached a minimum age of 23 years. Subjects may choose to withdraw from 
the study at any time and may request for their stored samples to be destroyed.  All 
withdrawals from the study will be documented and included in the annual report 
for continuing review by the IRB.   
• The PI will report to the IRB loss or destruction of data or samples when substantial 
numbers of samples/data records (more than 10) are unintentionally destroyed (for 
instance, by a freezer failure). Loss or destruction of samples saved only for tertiary 
outcomes or future unspecified research will not be routinely reported, unless such 
loss also entails a breach of the Privacy Act. 
b. Data and sample sharing plan 
(See Appendix F for Scientific Director Approval) 

46 
 
• This protocol is not subject to the Genomic Data Sharing policy as no genomic data 
will be collected. 
• Data will be submitted to the Clinical Trial Database (CTDB) for storage 
throughout data collection and to BRRIS at the conclusion of data collection.   
• A Data Sharing Plan for this study has been approved by the NICHD Scientific 
Director. See Appendix F, Data Sharing Plan for complete details. 
• Additionally: 
Data will be shared with Cogstate LTD and Britni Belcher, PhD, MPH (see 
Collaborating Researchers and Affiliated Institutions below). 
 
Data and samples may also be shared with collaborating laboratories at NIH or 
outside of NIH and/or submitted to additional NIH-designated repositories 
and databases if consent for sharing is obtained in the future with protocol 
amendment. 
 
Samples and data will be stripped of identifiers and will be coded (“de-identified”) 
or unlinked from an identifying code (“anonymized”). When coded data is 
shared, the key to the code will not be provided to collaborators, but will 
remain at NIH to permit the NIH research team to link clinically significant 
findings with a subject so that the subject can be informed and appropriate 
follow-up arranged if medically indicated.  Data and samples may be shared 
with investigators and institutions with an FWA or operating under the 
Declaration of Helsinki (DoH) and reported at the time of continuing review.  
Sharing with investigators without an FWA or not operating under the DoH 
will be submitted for prospective IRB approval.  Submissions to NIH-
sponsored or supported databases and repositories will be reported at the time 
of Continuing Review.  Submission to non-NIH sponsored or supported 
databases and repositories will be submitted for prospective IRB approval. 
 

47 
 
c. 
Collaborating Researchers and Affiliated Institutions: This paragraph may be 
amended in the future to describe any MTAs that are established to use samples from 
this study.  
• A technology transfer agreement with Cogstate LTD has been executed for this 
study.  This study will use Cogstate software as detailed throughout the protocol 
and in turn Cogstate LTD will be provided the following de-identified data:  
questionnaire performance, medical status (specifically any neurologic or 
psychologic diagnoses), ethnicity, level of education, yearly household income, and 
randomization assignment.  
• Extramural collaborator Britni Belcher, PhD, MPH will have continuous access to 
deidentified data (coded by subject number and otherwise without name, date of 
birth or other unique identifiers) throughout study to assist with data analyses.  
6. Additional Considerations 
a. 
Research with investigational drugs or devices 
 
Not Applicable. 
b. 
Gene therapy 
 
Not Applicable. 
7. Risks and Discomforts 
 
As with any research study, there may be risks, inconveniences, and discomforts that occur 
during the course of the study. There will be an effort make to minimize the risk and discomfort 
associated with the study.  
a. The physical examination by a health care provider will be conducted with the 
participant unclothed. While all efforts will be made to protect privacy, some participants 
may find this embarrassing.  
b. Non-invasive body composition methods such as waist circumference measurements 
will be done with a flexible tape measure; these measurements involve no risk and are not 
painful. However, some participants may find it embarrassing to stand in their underwear. 

48 
 
c. Total blood draw amount will remain within the NIH guideline of 5 mL/kg in a single 
day and 9.5 mL/kg every 8 weeks. Blood collection by venipuncture is associated with 
mild discomfort (which will be attenuated by offering to administer ELA max cream 
before venipuncture), and the possibility of localized bruising or extravasation. The risk 
of infection, phlebitis (a blood clot), or fainting is extremely small. An area of white or a 
red rash that usually goes away within a few hours may be seen after use of ELA max 
cream.   
d. The DXA scan may cause inconvenience of the time required and results in minor risk 
from radiation. DXA scanning involves radiation exposure for research purposes only.  
According to the manufacturer’s specifications, DXA scanning for body composition 
using the array beam delivers an effective dose of 0.003mrem total body radiation. This 
calculated value is known as the “effective dose” and is used to relate the dose received 
by each organ to a single value. This amount is well below the recommended radiation 
exposure for child volunteers (< 500 mrem per year). For comparison, the average person 
in the U.S. receives a radiation exposure of 0.3 rem (300 mrem)/yr from natural 
background sources, such as from the sun, outer space, and from radioactive materials 
that are found naturally in the earth’s air and soil. 
e. The exercise tests conducted in the study are low risk for participants without known 
heart disease. The major risk of exercise is that unsuspected heart disease could be 
uncovered. Even though participants will be examined before the study to try to rule out 
the presence of heart disease, it is possible to have undetected heart disease leading to 
exercise-related chest pain, an unusual heart beat rhythm (arrhythmia), or myocardial 
infarction (heart attack) with heart damage or death. All participants will undergo EKG 
and, if indicated by EKG results or physical examination, echocardiography before 
performing the maximal exercise test. We will monitor heart function as participants 
exercise, and stop the exercise if there are any signs of heart problems. A physician or 
Pediatric Advanced Life Support (PALS) (PALS) certified nurse practitioner will be 
readily available during these tests. In settings where apparently healthy adults undergo 
maximal exercise tests, the risk of death has been estimated to be between 0 and 3 
deaths/100,000 tests. Risk for death in otherwise healthy children will be lower than the 
rate cited for adults. Other risks of exercise include the possibility of slipping and falling 

49 
 
while exercising, muscle or joint soreness, and muscle fatigue caused by exercising that 
could last several days. Some individuals may exhibit mild skin irritation of the areas 
required for EKG lead placement. 
f. The Cognitive Tests and Mood Questionnaires involve no known risk. Participants 
may feel embarrassment or discomfort about answering questions about mood, anxiety, 
and performing cognitive tests. Although we expect this embarrassment or discomfort to 
be minimal, these tests and questionnaires do take time and may cause inconvenience. If 
there is slight discomfort due to the wording of some questions, the participant is not 
required to answer those particular questions. 
g. Using continuous glucose monitor (CGM) devices may cause some mild discomfort, as 
this device includes a subcutaneous insertion site through a minimally invasive needle 
stick.  The most common cited complication of CGM use is pain during insertion and 
skin irritation from adhesive material.  These concerns are infrequently reported.  For 
example, in randomizing approximately 100 adults and children to a CGM device, Diess 
et al. reported that 4 individuals withdrew CGM use citing pain, adhesive complication, 
or alarm complications.164 We will minimize these complications by using:  1.  Child Life 
services for education and distraction techniques in preparation for and during the 
insertion procedure, and 2.  Multiple adhesive techniques for skin application including 
skin preparation and various covering materials including skin tape and Tegaderm. 
Other than these concerns, CGM devices have never been reported to directly cause 
medical harm.  Zero significant adverse events such as infection were reported by Diess 
et al. (n = 108 randomized to CGM use).164 However, given that the insertion procedure 
requires breaking the dermis, there is always potential for introduction of infectious 
material into the body.  The procedure requires appropriate skin preparation and clean 
technique to prevent this.  For our study, only proficiently trained licensed providers will 
insert CGM devices to ensure proper technique.  Furthermore, given the superficial 
placement, if the skin and device preparation fail to prevent inoculation, prompt 
identification and removal of the device will allow for healing and clearing of a potential 
infection.  Our study will be providing close monitoring with daily evaluation of the 
CGM site by research study staff, making note of any signs or symptoms of infection or 
allergic reaction.  If any signs or symptoms develop, such as redness, swelling, skin 

50 
 
pealing, or drainage, or the subject reports irritation or discomfort to the study staff, a 
licensed provider will be notified, and the site will be removed if deemed appropriate.  
Replacement of a new device in a separate location will only be performed if removal 
was indicated due to skin irritation and will be executed by using new adhesive 
mechanisms.  If removal is indicated due to concern for infection, no additional CGM 
monitoring will be obtained on the participant in question.     
h. The study as a whole requires a significant time commitment on the part of study 
participants and their families. The screening visit will take approximately 6 hours. Each 
test visit will take approximately 3.5 hours. 
i. False Positives. Abnormal test results may cause anxiety and could lead to additional 
medical evaluations.  
j. MRS Scan.  This machine requires laying supine for up to approximately 120 minutes; 
therefore, there may be some discomfort or restlessness.  The child will be able to be in 
communication with their guardian and study team throughout the entire imaging in order 
to communicate significant distress or request to terminate the study. 
8. Subject Safety Monitoring 
 
Study participants will be examined at the screening visit and both test visits. At the beginning of 
each visit, participants will be asked whether they are experiencing any unusual symptoms, and 
measurements will be made of their vital signs (oral temperature and arm blood pressure). 
Stopping procedures for the VO2max test are outlined above. Heart rate will be monitored 
during test visit #6 and during walking bouts for participants randomized to “interrupted” 
condition (see above)..  If unanticipated serious adverse events occur during the course of the 
study, subjects will be withdrawn and we will promptly report them to the IRB. 
Criteria for withdrawal of subjects from study: 
 
Participants will be informed that they can withdraw from the study at any time. 
Noncompliance with study procedures may lead to subjects being withdrawn by Investigators. 
Other events that will result in study discontinuation include: 
a. Inability to complete treadmill fitness test. 
b. Inability to tolerate sitting (or sitting + walking) protocol. 

51 
 
c. The development of a severe psychiatric disorder. 
d. Any verified abnormal laboratory values or physical status that is not allowed by the 
study. 
9. Outcome Measures 
a. Primary outcome measure. The primary outcome is insulin iAUC obtained during the 
oral glucose tolerance test at the final experimental visit.  A difference in insulin demand, 
demonstrated by a difference in iAUC between groups, is hypothesized to occur with this 
intervention through increased non-insulin mediated glucose uptake.165, 166 A decrease in 
insulin demand is clinically relevant as it is a marker of improved carbohydrate 
metabolism and decreased risk of dysglycemia or the development of diabetes mellitus. 
b. Secondary outcomes are glucose iAUC obtained during the oral glucose tolerance test; 
mean iAUC as obtained from 24hr- continuous glucose monitoring, and Mean Amplitude 
Glycemic Excursion (MAGE) as obtained from 24hr- continuous glucose monitoring. 
Tertiary outcomes are cognitive function (working memory, inhibition, attention, and 
processing speed), mood, anxiety, daily physical activity (steps counts/day), and dietary 
intake at the final test meal.  Improved circulating glucose values as demonstrated by 
lower glucose iAUC would directly support our primary insulin outcome and would be 
clinically relevant as this result would indicate improved metabolism.  Additionally, 
MAGE has been demonstrated to be associated with metabolic control such that elevated 
MAGE is associated with worsening cardiometabolic outcomes in individuals with 
diabetes.167 
The executive function, mood and anxiety outcomes are being collected for independent 
objectives.  This group of measures is an important objective to provide information that 
will be necessary to know for feasibility of this intervention.  This intervention is poised 
to community application such as school class rooms.  Therefore, it is important to 
understand if executive function is adversely affected by the intervention as this would 
limit its feasibility for implementation.  Finally, all analyses will be performed within the 
subgroups of healthy weight and overweight subjects to evaluate for differential 
effectiveness based on weight status. 

52 
 
 
10. Statistical Analysis 
 
a. Analysis of data.  Prior to conducting the main model analysis, descriptive analyses will 
be conducted on all outcome and covariate to assess potential outliers and calculate 
summary statistics. If significant deviations from the assumed distribution (e.g.: normal) 
occur, then appropriate transformations will be applied to fulfill model assumptions. If 
transformations are not sufficient to meet model assumptions, then nonparametric tests 
will be used. Descriptive statistics will investigate correlations between variables at pre-
test and post-test to assess potential confounders prior to analyses.  
The parallel study design allows for the examination of between-individual comparisons 
of test effects and within-individual longitudinal test effects (i.e. repeated daily CGM 
metrics).  Significance level will be set at α= 0.05. Covariates will include: age, sex, 
race/ethnicity, baseline cognitive ability (for cognitive outcomes only), baseline 
homeostatic model of insulin resistance (HOMA-IR), body fat mass (except for subgroup 
analysis by weight status), and Tanner stage.  We will also evaluate for a weight status 
interaction (intervention x healthy weight / overweight).  
 
The following analyses will be performed: 
i. 
Participant characteristics will be summarized using means (standard deviations) and 
frequencies.  
ii. 
Baseline (time 0) values for participant characteristics, insulin, glucose, heart rate, 
and blood pressure between the groups will be compared using t-tests. 
iii. 
The primary analysis will be ANCOVA comparing children randomized to the two 
groups for insulin iAUC at the end-study oral glucose tolerance test, with age, sex, 
race/ethnicity, HOMA-IR, body fat mass, and Tanner stage as variables in the model. 
If all covariates are nonsignificant, unpaired t-tests will be reported for the differences 
in OGTT insulin iAUC by condition (prolonged vs interrupted sitting).  Similar 
analyses will be used to examine glucose iAUC, 5- or 6-day average 24hr CGM 
iAUC and 24hr MAGE. (Subjects may only have 5 days of CGM data if a device is 

53 
 
accidentally removed and replaced at the next visit or subject uses a medication that 
interacts with glucose readings such as acetaminophen-after which 24hrs of data will 
be omitted from analysis). Mixed models will also be used to assess the influence of 
prolonged vs. interrupted sitting on insulin and glucose iAUC over the 3 hours of 
observation. To evaluate differences in the dependent variables at each time point, 
planned comparisons of model-based mean differences between each time points will 
be calculated.  Additionally, mixed models will be used to assess condition influence 
on mean 24hr CGM glucose iAUC and 24hr MAGE. 
iv. 
ANCOVA or (as indicated above) t-tests will investigate differences in working 
memory, inhibition, attention, processing speed, mood, anxiety, and kcal intake 
between prolonged sitting and interrupted sitting at the post- test. 
v. 
Mixed models will also be used to assess the influence of prolonged vs. interrupted 
sitting on change in cognitive function, mood and anxiety (pre-post test scores) 
controlled for significant covariates.  Covariates to be considered include those listed 
above in addition to free living activity and sleep. 
vi. 
As a secondary analysis, all the analyses described above will be performed on the 
subgroups:  Healthy weight and overweight subjects.  
vii. 
Supplemental MRS Study:  Repeated measures ANOVA will investigate the change 
in IMCL content between baseline & pre-session (thus, evaluating for daily 
intervention effect) as well as immediately pre- and post-intervention session (thus, 
evaluating the immediate effect of intervention) by condition.  ANCOVA analyses 
will compare the change in IMCL content by condition with age, sex, race/ethnicity, 
HOMA-IR, body fat mass, and Tanner stage as variables in the model. 
 
b. Power Analysis 
Based on our preliminary published data,65 and our unpublished data in overweight 
youth, we expect a moderate to large (Cohen’s d = 0.8) effect size.  With an 80% power, 
α=0.05, our sample size requires evaluable data from 21 in each group; with our goal of 
subgroup analysis within healthy weight and overweight groups, we will require 84 
participants total (42 overweight and 42 healthy weight).  This is supported by previously 
reported adult data published by Dunstan et al. who identified the mean (standard 

54 
 
deviation) difference between the two groups was 191.0 (334.9) pmol/L yielding a 
moderate to large effect size.48 Taking into consideration more variability due to the 
parallel design (versus cross over designs used previously), we will randomize up to 120 
participants and estimate that at most 36 children will drop-out of the study before 
completion. We expect at least 84 participants to complete the protocol which is our 
target completion based on these analyses. 
11. Human Participant Protections 
a.  Rationale for subject selection 
This is a study to examine the effects of interrupting sitting on metabolic characteristics, 
executive function and attention, mood, anxiety, and dietary intake in children. Both 
healthy weight and overweight children will be studied because our pilot data suggest 
similar effects on insulin iAUC are seen. Pre-adolescent children were selected because 
the approach being studied is most suitable as a preventive measure that could be 
implemented in elementary or middle schools. This pilot study therefore has the potential 
to inform future intervention studies aimed at intervening on behaviors that promote 
pediatric obesity. Recruitment efforts will aim to enroll 50% girls, 50% boys, age 7 to 
<12. Participants from all socioeconomic backgrounds in the metropolitan Washington 
D.C. area will be invited to participate in the study. Children of all race/ethnic groups will 
be included. 
b. Equitability 
This study will provide equal opportunity to participants.  We will screen and enroll 
interested participants without concern of race, ethnicity, sex, disability, nationality, 
religion, handedness, or age (within inclusion criteria). 
c.  Justifications for exclusions: 
 
All exclusions are either based on identification of problems in the participant's 
physical or mental health, based on the ability of the participant to complete protocol 
studies, or are required for the goals of the study (e.g., age 7-11.99y). Individuals will be 
excluded if they are not proficient in English due to the availability of our mood and 

55 
 
anxiety testing questionnaires which are only available in English).  Identification of 
exclusions will lead to referral to an appropriate physician or mental health professional. 
d.   Justification for sensitive procedures:  N/A 
e.  Safeguards for vulnerable populations:  Children 
This study involves many minimal risk procedures: outpatient visits, physical 
examination, phlebotomy, anthropometric measurements, cognitive testing, exercise 
testing, and DXA scanning for child participants. Efforts will be made to schedule 
Clinical Center visits during school holidays and breaks to minimize absences. The 
protocol considers psychological as well as physical discomfort, and provision has been 
made for soliciting the written assent of children and the written permission of parents. 
Study staff will emphasize to participants that they are able to withdraw from the study at 
any time without penalty. To protect against the risk of embarrassment or discomfort, 
participants will be allowed to skip questionnaire items they do not wish to answer, or 
stop any study procedures. Written, active informed consent will be obtained from at 
least one parent/guardian (both will need to sign if otherwise required because, for 
example of joint custody), and assent will be obtained from children.  
Every effort will be made to maintain the confidentiality of participant medical records 
for this study. Only group information without personal identifiers will be included when 
submitting manuscripts for publication. All information gathered for the purposes of this 
study will be treated as a medical record with the same degree of confidentiality.  
 
12. Risks/Benefits Analysis 
a. Known Potential Benefits: 
There are no expected benefits to participants.  
b. Known Potential Risks 
Potential risks and discomforts are described in the consent form and in Section 7. These 
include discomfort or bruising during intravenous catheter placement and blood draws, 

56 
 
nausea during glucose tolerance testing, and the risks of walking on a treadmill (such as 
spraining an ankle) and of undergoing MRI (claustrophobia), In the literature, there are 
reports of healthy children who have undergone each of these tests in IRB-approved 
studies. Individually, all the procedures proposed as part of this protocol have been 
considered to represent minimal risk. For example, a panel convened in 1993 by the 
Secretary of Health found that placement of two intravenous catheters, administration of 
insulin and dextrose for the purpose of inducing mild hypoglycemia, and monitoring of 
blood glucose measurements every 5 minutes for many hours were of "minimal risk." 
The investigators have further taken many steps to minimize potential discomfort, for 
instance by administration of ELA max cream before blood is drawn. Children also 
routinely undergo venipuncture, which in children not infrequently requires multiple 
sticks to obtain samples. Furthermore, children are routinely required to complete 
cognitive tests during school. We believe the studies outlined in this protocol are 
reasonably commensurate with those children may be exposed to in their actual or 
expected medical, dental, psychological, social, or educational experiences. We therefore 
believe that the minimal risks of this study are reasonable in relation to anticipated 
benefit.  
 
13. Consent Documents and Process 
 
a. 
Designation of those obtaining consent 
 
Study investigators designated as able to obtain consent in this protocol document will 
obtain informed consent from parents or guardians of study subjects.  Written assent 
will be also obtained from the minor subjects of this study.  See Appendices C & D for 
consent and assent documents. 
b. 
Consent procedures  
Each participant will receive a written explanation of the purposes, procedures, and 
potential hazards of the study. Communication of this information and of the 
participant’s assent as well as the consent of a parent or guardian will be documented in 
the medical record. Written, active informed consent will be obtained in-person during 

57 
 
the screening visit from a parent/guardian, and assent will be obtained from youth by 
the PI or designated associate investigators. All participants will be informed of their 
right to withdraw from the study. The purpose of the project, all testing procedures, and 
study components, and possible risks and inconveniences will be described in detail in 
language understandable to the parent(s)/guardian(s). The investigator will also explain 
the study to the minor who is of a younger age and level of understanding. Sufficient 
time and opportunity will be given for discussion of the research as well as to answer 
any questions they may have, taking care to minimize or eliminate the perception of 
coercion or undue influence. When the NIH informed consent and assent forms are 
signed, parents or guardians will be given a signed copy of the consent and assent form 
for their records. In the unlikely event that the parents/guardians of protocol-eligible 
subjects cannot travel to the NIH Clinical Center for a considerable period of time, the 
PI or other protocol staff designated to obtain consent will obtain informed consent by a 
telephone conversation. Consent may be obtained via telephone and/or another 
electronic process, rather than in person. In such cases, the written signed consent will 
be faxed and/or mailed and made part of the patients' NIH records. No research 
procedures will commence until the investigator has confirmed that written, legally 
effective consent has been obtained. In general, Investigators obtaining consent 
(whether in person or over the telephone) will be encouraged to have a second person 
(who is not obtaining informed consent) present during the entire informed consent 
discussion and, if applicable, the signature process. Investigators will also document the 
consent process in a note placed in the research record for all consent discussions. 
 
As noted above, given the minimal risk of the study, the consent of one parent will be 
considered sufficient for protocol participation except in cases where two signatures are 
required (for instance because of shared medical decision-making explicated in a 
divorce decree). 
c. 
Non-English–Speaking Participants 
If a child who speaks English has a parent who is found not to be English-speaking is 
eligible for enrollment, the parent will be provided with the CC Short Written Consent 
Form for Non-English Speaking Research Participants and a verbal explanation of the 

58 
 
purpose, procedures and risks of the study as described in MAS Policy M77-2, NIH 
HRPP SOP 12 and 45 CFR 46.117(b)(2). The IRB-approved English consent form will 
serve as basis for the verbal explanation of the study. All NIH policies on utilizing the 
“short form” consent process, including having a witness during the "short form" 
consent process, will be followed.  
d. 
Consent documents 
The submitted consent form contains all required elements for consent. One consent 
form, for parents/guardians to sign for their minor children, and one assent form, for 
minor children, are submitted with this protocol.  See Appendices C & D. 
 
14. Data and Safety Monitoring 
a. 
Data and safety monitor 
 
For this un-blinded, minimal risk trial, we propose that the PI will be sufficient to 
monitor the study. 
 
b. 
Data and safety monitoring plan for Non-IND 
The following monitoring plan will be implemented to ensure the protocol data are in 
compliance with Good Clinical Practice (GCP), NICHD Institutional Review Board 
(IRB) and Federal regulations. This will ensure the validity, accuracy and integrity of 
the data. 
 
1.  Study Staff Responsibilities  
Dr. Jack Yanovski, Principal Investigator, will be responsible for all aspects of the study. 
Some responsibilities may be delegated to other associate investigators as detailed in the 
Study Personnel Sheet & Delegation of Responsibility Log.  
 
2.  Source Documentation and Case Report Forms 
Jack A. Yanovski, MD, PhD is responsible for coordinating data collection and will 
review the data for accuracy and completeness within seven days of each subject visit.  

59 
 
The PI along with study associate investigators will conduct the monitoring. Patient 
consent documents, primary outcome and safety laboratory results, and diagnostic test results 
will be monitored for accuracy, correct dating, and agreement between case report forms and 
source documents. As case report forms are entered electronically into the NICHD Clinical 
Trials Database, the computer system contains logs indicating changes made and the 
circumstances leading to these changes.  
The medical records of active subjects (defined as subjects undergoing study procedures 
in the current year) will be monitored quarterly or more frequently as required.  Any major 
findings will be summarized in writing and reported to the NICHD Institutional Review Board, if 
indicated. Investigator credentials, training records, and the delegation of responsibility log will 
also be reviewed on an annual basis. 
3.  IRB and DSMC Documentation  
All IRB documentation can be found in iRIS. The Principal Investigator, Jack Yanovski, 
MD, PHD, is responsible for maintaining IRB correspondence related to this protocol, including 
records of all reviews of the study and submissions to the IRB. This protocol does not meet the 
criteria as outlined in the NIH HRPP SOP 17 for data to be submitted to a data safety monitoring 
committee.   The PI and IRB can provide adequate oversight for this open trial. 
Clinical Trial Studies (non IND/IDE) will have random audits performed by the NICHD 
Office of the Clinical Director: 
https://science.nichd.nih.gov/confluence/display/ocd/Protocol+Navigation). 
4.  FDA Documentation  
Not Applicable – no IND. 
5. Study Completion 
Upon completion of the study Jack A. Yanovski, MD, PhD will retain possession of the 
electronic Protocol Binder in a secure location. HHS regulations require that subjects’ records be 
maintained for at least three years after completion of a study. Since subjects of this investigation 
include children age 7-11.9 years, even longer record retention is required. All records will be 
retained until all participants have reached age 23 years. 
6. Data safety and monitoring board (DSMB) 
Because there is no blinded component to these studies, the studies do not involve 
administration of any pharmaceutical agents, and the procedures are of minimal risk, we propose 

60 
 
that a formal DSMB is not required, and that the investigators and IRB can adequately supervise 
this study. All adverse events will be documented and reported to the NICHD IRB as described 
above in ‘Protocol Monitoring Plan.’ We expect that the risk for an adverse event will be within 
the “Rare but serious (Event Rate < 1%)” category. 
 
15. Quality Assurance 
a. 
Quality assurance monitor 
Jack A. Yanovski, MD, PhD is responsible for coordinating data collection and will 
review the data for accuracy and completeness within seven days of each subject visit. 
The NICHD Standard Operating Procedure for Intramural Clinical Protocol Monitoring 
(rev. 5.20.2015) will be followed. 
 
b. 
Quality assurance plan 
 
Quality assurance will be executed in accordance with the NICHD quality assurance 
plan. Adverse events and protocol deviations will be reviewed and recorded on a 
weekly basis at the SGO protocols review meeting. 
Protocol-specific monitoring will include: 
Review of biochemical results from OGTT testing within 72 hours of test. 
Daily data retrieval from continuous glucose monitoring devices. 
Weekly data retrieval from continuous actigraph and heart rate monitoring devices. 
Weekly data retrieval from the NIH Toolbox Assessment center for cognitive 
measure outcomes. 
16. Reporting of Unanticipated Problems, Adverse Events and 
Protocol Deviations 
 
Event Characterization and Reporting to the IRB and Clinical Director (CD): 
Unanticipated problems, non-compliance, and other reportable events will be reported to the NIH 
IRB as per Policy 801 (“Reporting Research Events.”). All reportable events occurring during 

61 
 
the study, including those observed by or reported to the research team, will be recorded. 
Reportable Events will be reported by the PI verbally and in writing as soon as possible. but not 
more than 7 days after the PI first learns of the event. Deaths will be reported to the Clinical 
Director within 24 hours after the PI first learns of the event. All adverse events (serious and 
non-serious, expected and unexpected) will be reported annually to the IRB for review. 
Electronic data sheets or summary language will be supplied for IRB review of adverse event 
reports at continuing reviews. 
The Principal Investigator is responsible for detecting, documenting, and reporting 
unanticipated problems, adverse events (AEs), including serious adverse events (SAEs), and 
deviations in accordance with NIH policy, IRB requirements, and federal regulations.  
17. Alternatives to Participation  
The current proposal is a physiological study that does not treat any known condition; as 
a result, there are no alternatives other than non-participation. Participants and their families will 
be informed that they will not receive treatment as a result of study participation. For participants 
found to have medical conditions, treatment referrals will be provided. 
18. Privacy 
All research activities will be conducted in as private a setting as possible.  This will be most 
noteworthy during the history and physical examination will be conducted in a private room in 
the pediatric day hospital. 
19. Confidentiality 
 
• Samples and data will be stored using codes that we assign. Data will be kept in 
password-protected computers and on the secured data server hosted by the Section 
on Growth and Obesity as described above.  Samples will be kept in locked storage. 
Only study investigators will have access to the samples and data.   
• De-identified results will be posted on clinicaltrials.gov 

62 
 
20. Conflict of Interest 
 
a. 
Distribution of NIH guidelines 
• NIH guidelines on conflict of interest have been distributed to all investigators.   
b. 
Conflict of interest  
This is a covered protocol because of the research agreement with Cogstate LTD. 
There are no conflicts-of-interest to report for NIH investigators.  Non-NIH 
investigators will abide by the conflict-of-interest policies of their own institutions. 
c.  Role of a commercial company or sponsor  
Cogstate supplies software for collection of cognitive data during the study and will 
have access to some data under the terms of the signed agreement. The company 
had no role in the study design and will have no role in the decision to publish. 
21. Technology Transfer 
There is a technical transfer agreement negotiated with Cogstate LTD.  See Collaborating 
Researchers and Affiliated Institutions under Management of Data and Samples above.  
22. Research and Travel Compensation  
Volunteers will be compensated for time and research-related inconveniences as follows:  
1. Complete screening visit: $100 
2. Complete outpatient test visits days #1-5: $100/visit (5 visits; $500 total) 
3. Complete final testing visit, day #6:  $150 
4. Complete CGM wear: $20/wear (6 days; $120 total) 
5. Complete wrist activity monitor wear:  $10/day (6 days; $60 total) 
(Study total $930) 
 
Volunteers taking part in the MRS Supplemental arm of the study will be compensated 
for time and research-related inconveniences as follows: 
1. Complete baseline MRS scan: $100 
2. Complete follow up scan #1 (immediately before testing session): $75 
3. Complete follow up scan #2 (immediately after testing session): $75 
(Study Total $250) 
 
 
Compensation will be prorated for parts completed if subjects do not complete the 
study as guided by the reimbursement plan above. 
No additional incentives will be provided.  
Payment will be provided in the name of child in check form. 

63 
 
Payment will be issues within 7 days after completion of last study visit. 
Travel/lodging compensation.  Local travel may be covered to facilitate the daily 
visits required. 
• No escort fee will be provided. 
• NIH employees or staff who wish to have their child participate during work hours 
must have permission from their supervisor. NIH employees or staff must either 
participate outside of work hours or take leave in order to receive compensation. 
 
 
 
 
 

64 
 
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