Strategies to Moderate Energy Intake for the Prevention of Obesity in Children
Phase: NA
Conditions: Feeding Behavior
Sponsor: Penn State University

Effect of Varying Proportions of Low- and High-Energy-
Dense Foods Over 5 Days in Preschool Children 
 
ClinicalTrials.gov Protocol ID: ChildFood503 
ClinicalTrials.gov ID Number: NCT03242863 
 
 
Principal Investigator: Dr. Barbara J. Rolls 
Sponsor: The Pennsylvania State University 
 
 
Grant Title: Strategies to Moderate Energy Intake 
for the Prevention of Obesity in Children 
Grant Number: R01DK082580 
Funded by: National Institute of Diabetes and Digestive 
and Kidney Diseases 
 
 
Institutional Review Board 
The Pennsylvania State University 
IRB ID: Study00001832 
Initial Approval Date: 28 January 2015 
IRB Protocol Document version 0.19 
Approval Date: 31 August 2018

 
Table of Contents 
 
1.0 
Objectives 
  1 
2.0 
Background 
  2 
3.0 
Inclusion and Exclusion Criteria 
  3 
4.0 
Recruitment Methods 
  3 
5.0 
Consent Process and Documentation 
  4 
6.0 
Study Design and Procedures 
  6 
7.0 
Statistical Plan 
  9 
8.0 
Confidentiality, Privacy and Data Management 
10 
9.0 
Data and Safety Monitoring Plan 
12 
10.0 Risks 
12 
11.0 Potential Benefits to Subjects and Others 
12 
12.0 Sharing Results with Subjects 
12 
13.0 Economic Burden to Subjects 
12 
14.0 Other Approvals 
13 
15.0 Adverse Event Reporting 
13 
16.0 Study Monitoring, Auditing and Inspecting 
13 
17.0 References 
14 
 

1 
 
1.0 
Objectives 
 
1.1 
Study Objectives 
 
Aim: To determine the effect on food and energy intake of varying the 
proportion of lower-energy-dense vegetables and fruits in meals served 
over five days to preschool children. 
 
Hypotheses: 
 
1. When low-energy-dense vegetables and fruits are added to children’s 
meals over five days (Addition condition): 
a. Daily energy intake will increase compared to the Baseline condition 
b. Daily vegetable and fruit intake will increase compared to the 
Baseline condition  
c. Daily food and beverage intake will increase compared to the 
Baseline condition  
d. Daily food energy density will decrease compared to the Baseline 
condition 
 
2. When low-energy-dense vegetables and fruits are substituted for foods 
higher in energy density in children’s meals over five days (Substitution 
condition): 
a. Daily energy intake will  
i. decrease compared to the Baseline condition 
ii. decrease compared to the Addition condition 
b. Daily vegetable and fruit intake will 
i. increase compared to the Baseline condition  
ii. increase compared to the Addition condition  
c. Daily food and beverage intake will 
i. not differ significantly from the Baseline condition 
ii. decrease compared to the Addition condition 
d. Daily food energy density will 
i. decrease compared to the Baseline condition 
ii. decrease compared to the Addition condition 
 
3. Across all three conditions of different proportions of low-energy-dense 
vegetables and fruits, daily intakes of vegetables, fruit, and energy will 
begin to converge over the five-day period 

2 
 
1.2 
Primary Study Endpoints 
 
1. Differences in food and beverage intake by energy [Time Frame: Day 
1-5 in Weeks 1, 2, and 3] 
Energy intake in kcal 
2. Differences in food and beverage intake by weight [Time Frame: Day 
1-5 in Weeks 1, 2, and 3] 
Weight consumed in grams (g) 
 
1.3 
Secondary Study Endpoints 
 
1. Differences in food and beverage intake by energy density [Time 
Frame: Day 1-5 in Weeks 1, 2, and 3] 
Energy density in kcal/g 
 
 
2.0 
Background  
 
2.1 
Scientific Background and Gaps 
 
It has been proposed that preschool children are in a formative stage 
during which the ability to regulate energy intake is being replaced by 
eating in response to environmental food cues. In recent studies, we have 
found that increases in both food portion size (g) and energy density 
(kcal/g) have been robust in promoting excess energy intake in preschool 
children [1,2]. These results challenge the proposition that preschool 
children show self-regulatory behavior by altering their energy intake when 
it is perturbed by variations in the available foods. 
 
At present no controlled studies have been conducted over a sufficient 
duration to determine whether preschool children adjust for energy 
imbalances stemming from changes in the proportions of foods lower and 
higher in energy density served at meals. 
 
2.2 
Previous Data 
 
N/A 
 
2.3 
Study Rationale 
 
This study will provide a better understanding of the regulation of energy 
intake in preschool children over multiple days by determining how intake 
is affected by changing the proportions of foods lower and higher in 
energy density that are served at meals.  

3 
 
3.0 
Inclusion and Exclusion Criteria 
 
3.1 
Inclusion Criteria 
1. Children who are enrolled in a participating childcare center 
2. Children who are between the ages of 3 and 5 years old at the time of 
enrollment 
3. Parents of children enrolled in the study who are 18 years or older 
4. Teacher in a classroom with enrolled children who are 18 years or 
older 
 
3.2 
Exclusion Criteria 
1. Allergy to any of the foods served 
2. Children whose diets exclude any of the foods served (e.g. vegan, 
vegetarian, gluten-free) 
3. Children who are not planning to be present for all three study weeks 
(e.g. planned vacation during a study week)  
 
3.3 
Early Withdrawal of Subjects 
 
3.3.1 Criteria for removal from study 
 
Subjects will be removed from the study if they miss three or more 
days in one study week or two meals per day for three or more 
days in one study week. 
 
Subjects will also be removed from the study if they repeatedly fail 
to follow study protocol. 
 
All participation is voluntary and a child can refuse a session and 
parents may withdraw from the study at any time. 
 
3.3.2 Follow-up for withdrawn subjects 
 
N/A 
 
 
4.0 
Recruitment Methods 
 
4.1 
Identification of subjects 
 
We will recruit through a parent letter placed in each child's mailbox. Any 
children enrolled in the childcare center, along with their parents, may 
participate. A trained staff member will be at the childcare center at the 
time of child drop off and/or pick up to explain this study. 
 

4 
 
Teachers will be recruited verbally in the classrooms of participating 
children. 
 
4.2 
Recruitment process 
 
We will recruit through a parent letter placed in each child's mailbox. A 
member of our lab approved to do recruitment and consenting of subjects 
will be available in the center for an evening during child pick up to answer 
any questions parents may have. 
 
Teachers in classrooms will be asked to participate verbally by study staff. 
 
4.3 
Recruitment materials 
 
Parents will receive a letter in their child’s mailbox that will describe the 
study. The consent form will be included with the letter. 
 
Teachers in classrooms will be asked to participate verbally by study staff.  
A consent form will then be reviewed and signed by each teacher willing to 
participate. 
 
4.4 
Eligibility/screening of subjects 
 
Children with allergies to foods served will not be eligible to participate in 
this study. Screening questions regarding food allergies and intolerances 
will be included with the consent form. 
 
 
5.0 
Consent Process and Documentation  
 
5.1 
Consent Process  
Participation in this study is voluntary. 
 
5.1.1 Obtaining Informed Consent 
 
5.1.1.1 
Timing and Location of Consent 
Consent will be signed by the parent. Consent will be 
obtained at the childcare center with a trained member of 
the lab staff present. 
 
Teachers will consent prior to the first study week in their 
classroom at the childcare center to fill out questionnaires 
about children enrolled. 
 

5 
 
 
5.1.1.2 
Coercion or Undue Influence during Consent 
 
Parents will be given consent form to take home to 
review on their own and can decide if they want their 
child to participate. 
Teachers will be given the opportunity to review the 
consent from on their own and can decide if they wish to 
participate. 
 
5.1.2 Waiver or alteration of the informed consent requirement 
 
N/A 
 
5.2 
Consent Documentation 
 
5.2.1 Written Documentation of Consent 
 
Participation in this study is voluntary. 
 
Participants’ parents will sign the consent form. Two copies of the 
consent form will be given, one to be signed and returned to study 
staff, one to be kept for parents’ records. 
 
Teachers will sign a separate consent form for their participation 
and will be given a copy for their own records. 
 
5.2.2 Waiver of Documentation of Consent 
 
For addendum consent – taking the packed meal(s) implies 
consent. 
 
5.3 
Consent – Other Considerations  
 
5.3.1 Non-English Speaking Subjects 
 
It is not anticipated that there will be any non-English speaking or 
illiterate subjects. 
 
5.3.2 Cognitively Impaired Adults 
 
5.3.2.1 
Capability of Providing Consent 
 
N/A 
 

6 
 
5.3.2.2 
Adults Unable To Consent 
 
N/A 
 
5.3.2.3 
Assent 
 
N/A 
 
5.3.3 Subjects who are not yet adults (infants, children, teenagers)  
 
5.3.3.1 
Parental Permission 
 
Parents will sign consent for their child to participate. Two 
copies of the consent form will be given, one to be signed 
and returned to study staff, one to be kept for parents’ 
records. 
 
5.3.3.2 
Assent 
A child can refuse to eat any meal if he/she chooses. 
Before height and weight is measured or the Tasting 
Game occurs, children are asked if they want to do it and 
can refuse to participate. 
 
 
6.0 
Study Design and Procedures 
 
6.1 
Study Design  
 
This study will have a within-subjects crossover design with repeated 
measures across five days of meals and three experimental conditions. 
The investigators will vary the proportions of low-energy-dense vegetables 
and fruits served at meals by either addition or substitution. In the three 
experimental conditions, the same foods will be served - only the amounts 
and proportions of foods will be varied. In the Baseline condition, typical 
proportions of age-appropriate foods will be served. In the Addition 
condition, the portion sizes of low-energy-dense vegetables and fruits will 
be increased, and in the Substitution condition, the portions of low-energy-
dense vegetables and fruits will be increased by replacing an equivalent 
weight of foods higher in energy density. The order of the conditions 
across the three experimental periods, as well as the order of the five 
menus within each five-day period, will be counterbalanced by classroom. 
The three five-day experimental periods will be separated by a two-week 
washout period. All menus in all conditions will meet the minimum 
requirements of the United State Department of Agriculture (USDA) Child 
and Adult Care Food Program (CACFP) [3]. 
 

7 
 
6.2 
Study Procedures 
 
1. Over five consecutive days, enrolled children will be served breakfast, 
lunch, dinner, and snacks of foods commonly served in the childcare 
center following their regular meal and snack schedules - menus were 
developed from the menus at the childcare centers to be as similar as 
possible to the foods that children are regularly served. Milk or water 
will be served as beverages. Meals will be served at tables of three to 
six children and one adult, which is a standard practice at the childcare 
centers. The adults at the table will be instructed not to discuss the 
food or to encourage the children to eat. There will be three conditions, 
with changes only in the proportion of lower-energy-dense vegetables 
and fruits at the meals. Each participant will be served an individual 
pre-weighed meal. All food and beverage items will be weighed with a 
food scale before and after meals and snacks to within 1 gram in order 
to determine the amount consumed. Participants will be told they can 
eat as much or as little of the foods as they would like. Trained 
observers (research personnel) will document food and beverage 
spillage and any comments made about the food. The children will be 
provided with an evening snack pre-packaged for consumption at 
home. Any leftover food from the evening snack will be returned to the 
childcare center the following day to be weighed. 
2. In the event of extenuating circumstances (e.g. unexpected childcare 
center closure, inclement weather) the researchers may decide to pack 
out a meal, or meals, which can be consumed outside of the childcare 
center. Participants will be notified in advance if this should occur. Any 
leftover food from a packed meal, or meals, will be returned to the 
childcare center the following day to be weighed. Applicable 
instructions for serving or reheating will be provided. 
3. Parents will be asked not to serve any additional food or beverages to 
their child apart from those provided by the study and water. If the 
participant deviates from the procedure and consumes extra food, the 
estimated food and beverage consumption will be requested.  
4. Physical activity levels will be measured over the five consecutive days 
using triaxial accelerometers. The accelerometers will be worn while 
the children are present at the childcare center (from before breakfast 
to after dinner). Research staff will attach a device to the child’s waist 
upon arrival at the center each morning and remove the device upon 
the child’s departure from the center each evening.  
5. On the 16th visit to the childcare center, children will have their height 
and weight measured by a trained staff member of the lab. Body 
weight will be measured in triplicate using a portable digital scale. 
Height will be measured in triplicate using a portable stadiometer. The 
children will also complete the Tasting Game. They will be given small 
samples of foods served in the study and asked to point to a cartoon 
face that represents their liking of the food on a scale of yucky to 

8 
 
yummy. Children will also look at three plates (one from each 
condition) from two meals and be asked which they would prefer. 
6. Parents will be asked to complete an Evening Report Form, 
Background Questionnaire, the Children’s Eating Behavior 
Questionnaire, the Caregiver’s Feeding Style Questionnaire, and the 
Child Feeding Questionnaire. The Evening Report Form will be 
completed at the end of each study day and returned each morning to 
research personnel. All questionnaires (the Background Questionnaire, 
Children’s Eating Behavior Questionnaire, Caregiver’s Feeding Style 
Questionnaire and Child Feeding Questionnaire) will be administered 
during the last five days of the study. All questionnaires will be 
completed via pencil and paper. 
7. Photos of children eating, participating in the tasting game, and having 
their height and weight measured may be taken. 
8. There will be a minimum of a one-week break between study weeks. 
There will be a four- to six-week wash-out period between running this 
and any previous experiments at each childcare center.  
9. Approved lab staff (on this IRB application) will email parents the 
Friday before a study week to remind parents of the start of the study. 
10. Teachers will be asked to answer two questionnaires (Caregiver’s 
Feeding Style Questionnaire, Children’s Eating Behavior 
Questionnaire) per child in their classroom. Teachers will only 
complete questionnaires about children who are enrolled in the study. 
This questionnaire will be administered by paper and pencil. Teachers 
will be verbally told by study personnel which child to answer each 
questionnaire about. 
11. Data from questionnaires completed by teachers will be linked to child 
participants by a three-digit number and letter only. 
 
6.3 
Duration of Participation 
 
The duration of the study will be five consecutive days, and each 
participant will be asked to complete three of these five-day periods. 
Within each day, each meal and snack will take approximately 30 minutes 
to complete. The estimated time required by the participant each day is 
2.5 hrs. The estimated total time required for all of the meals is 37.5 hours. 
The height and weight measurements and the Tasting Game on the final 
visit after the 15 days are completed will require an additional hour of time. 
For the parents, another 30 minutes will be requested to complete the 
questionnaires. The total time commitment for the study is 39 hours. 
Teachers will spend approximately 20 minutes per questionnaire. 
 
 
 
 

9 
 
7.0 
Statistical Plan 
 
7.1 
Sample size determination 
 
The sample size for the study was based on the statistical power to detect 
differences in energy intake between the Baseline and Substitution 
conditions across study days. The sample size was estimated from a 
linear mixed model and an approximation technique [4] using exemplary 
data sets based on our previous five-day studies in preschool children 
[1,2]. We assumed that the minimum clinically significant difference in 
energy intake across days was 50 kcal, which is about 5% of daily intake 
for a typical preschool child. To detect this change over time with a Type 1 
error rate of 5% and power of 90%, it was determined that 48 participants 
would be required. To allow for withdrawals and non-compliance to the 
protocol, 55 to 60 children will be enrolled. 
 
7.2 
Statistical methods 
 
The study hypotheses will be assessed using linear mixed models with 
repeated measures. Differences in mean outcomes will be tested across 
the three experimental conditions of the proportion of vegetables and fruits 
served in meals, as well as across the five days of intake. 
 
Hypotheses 1 and 2 test whether variations in the proportion of low- and 
high-energy-dense foods influence the mean daily outcomes of energy 
intake, food and beverage intake, and energy density. To test these 
hypotheses, the fixed factors in the models will be condition, study week, 
study day, menu, childcare center, classroom, and sex. Interactions 
between these factors will be tested and removed from the model if not 
significant and participants will be treated as a random effect. The Tukey-
Kramer method will be used to adjust for multiple pairwise comparisons 
between means. The energy content of parentally reported non-study food 
consumption will be estimated by research staff. Daily energy intake will 
be analyzed both with and without the non-study items. 
 
Hypothesis 3, concerning whether daily energy intake in the three 
conditions converges over each five-day period, tests whether regulation 
of energy intake occurs over time in response to variations in the 
proportion of low- and high-energy-dense foods served. To test this 
hypothesis, a random coefficients model will be used. The trajectories of 
intake over time will be allowed to vary randomly across subjects; thus, 
they will be modeled separately for each child. Time (study day) will be 
treated as a continuous covariate in the model and polynomial factors of 
time (linear and quadratic coefficients) will be tested as both fixed and 
random effects to determine the shape of the intake trajectory. The 
intercept of the trajectory will reflect the magnitude of daily intake at Day 1 

10 
 
and the linear coefficient will represent the rate of change in intake (slope) 
across time after Day 1. The quadratic coefficient, if significant, will 
represent the rate of acceleration or deceleration in intake (curvature) 
across time after Day 1.  
 
 
It has been predetermined that children who have incomplete intake 
measures on ≥3 days in all 3 conditions will be excluded from analysis. It 
has also been predetermined that children who eat >95% of the food 
weight served on at least 1 day will be classified as “plate cleaners”, and 
their influence on the results will be tested.  
 
Analysis of covariance with linear mixed models will be used to test the 
influence on these outcomes of individual subject characteristics such as 
age, body mass index, and daily level of physical activity. Independent-
samples t-tests will be used to assess differences in subject 
characteristics by sex and weight status (normal weight or 
overweight/obesity). Results will be considered significant at p < 0.05. All 
data will be analyzed using SAS software (SAS 9.4; SAS Institute, Inc.). 
 
 
8.0 
Confidentiality, Privacy and Data Management  
 
8.1 
Confidentiality 
 
We will take photos of the children having a test meal and may use them 
for poster or oral presentations at scientific meetings. Parents will check 
on their consent form if they agree to have their child’s photo taken. All 
images will be stored on a password-protected network drive that only 
those on this application have access to. Images will be stored with a 
generic title and will not be labeled with any identifiable information. The 
images will be deleted from the drive within three years of completion of 
the study. 
 
 
8.1.1 Identifiers associated with data and/or specimens 
 
Subjects will only be identified by a three-digit subject number, dot 
color, and a letter of the alphabet. 
Teachers will not have their own identifiers. 
 
8.1.1.1 
Use of Codes, Master List 
 
The master list that links subject numbers to personal 
information will be stored in a locked closet in the Lab 
Manager's office. Only those listed on this application will 
have access to it. The master list will be destroyed upon 

11 
 
publication of the study or five years after the close of the 
study, whichever comes first. 
 
8.1.2 Storage of Data and/or Specimens 
 
Paper data will be stored in a locked cabinet in a locked closet in 
the Lab Manager’s office. Data on network drives will have no 
identifiable information. All computers and network drives are 
password protected. 
 
Paper and computer records of data will be stored indefinitely with 
identifiers removed. Paper records will be stored in a locked filing 
cabinet in the Lab Manager’s office. Computer records will be 
stored on a password-protected network drive that only those on 
this application have access to. 
 
This study will be issued a Certificate of Confidentiality. 
Researchers will not disclose or provide any identifiable information 
without the subject’s prior consent or where permitted according to 
NIH’s Policy on Issuing Certificates of Confidentiality. 
 
8.1.3 Access to Data and/or Specimens 
 
Only those listed on this application will have access to the data. 
 
8.1.4 Transferring Data and/or Specimens 
 
The researchers do not plan to release identifiable information 
collected in the study. However, if researchers consider releasing 
identifiable information in the future – the individual or institution 
receiving the identifiable information will be made aware they are 
also subject to the requirements of subsection 301(d) of the Public 
Health Service Act. 
 
8.2 
Privacy 
 
No personal information other than first name of the child, and telephone 
number and email address of the parent, is accessible to research staff. 
Subjects remain anonymous except for study number and letter. Only 
senior staff members listed on this IRB application will have access to 
personal information such as address, age, weight. 
 
Data on food consumption is collected in a separate room where only 
study staff is present. Heights and weights for children are collected in a 
private area of the child’s classroom. 
 

12 
 
9.0 
Data and Safety Monitoring Plan 
 
The proposed study poses minimal risk to participants, since the procedures 
involve little change from the usual meals and activities at the childcare centers.  
 
 
10.0 Risks 
 
There are minimal risks associated with participation in this project. The risks are 
no greater than those associated with the children’s usual attendance and 
consumption of meals at the childcare center and at home, which include the risk 
of food allergy, food-borne illness, and choking on food.  
 
The amounts of food and beverages served in all conditions meet or exceed 
minimum requirements for calorie and nutrient content for this age group as 
specified by the USDA and CACFP. 
 
There are no potential risks for teacher participants. 
 
 
11.0 Potential Benefits to Subjects and Others 
 
11.1 Potential Benefits to Subjects 
 
There are no direct initial benefits to subjects participating in the proposed 
study. 
 
11.2 Potential Benefits to Others 
 
The research will benefit society by advancing understanding of children’s 
eating behavior and identifying meal-related strategies for the prevention 
of obesity in children. 
 
 
12.0 Sharing Results with Subjects 
 
No results will be shared with the subjects. 
 
 
13.0 Economic Burden to Subjects 
 
13.1 Costs  
 
N/A 
 

13 
 
13.2 Compensation for research-related injury 
 
It is the policy of the institution to provide neither financial compensation 
nor free medical treatment for research-related injury. In the event of injury 
resulting from this research, medical treatment is available but will be 
provided at the usual charge. Costs for the treatment of research-related 
injuries will be charged to subjects or their insurance carriers.  
 
 
14.0 Other Approvals 
 
Local childcare centers have given permission for the conduct of this research in 
their facilities. Letters of permissions are on file with the Institutional Review 
Board. 
 
 
15.0 Adverse Event Reporting 
 
15.1 Reporting Adverse Reactions and Unanticipated Problems to the 
Responsible IRB 
 
In accordance with applicable policies of The Pennsylvania State 
University Institutional Review Board (IRB), the investigator will report, to 
the IRB, any observed or reported harm (adverse event) experienced by a 
subject or other individual, which in the opinion of the investigator is 
determined to be (1) unexpected; and (2) probably related to the research 
procedures. Harms (adverse events) will be submitted to the IRB in 
accordance with the IRB policies and procedures. 
 
15.2 Auditing and Inspecting 
 
The investigator will permit study-related monitoring, audits, and 
inspections by the Penn State quality assurance program office(s), IRB, 
the sponsor, and government regulatory bodies, of all study related 
documents (e.g. source documents, regulatory documents, data collection 
instruments, study data). The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory). 
 
 
16.0 Study Monitoring, Auditing and Inspecting 
 
16.1 Auditing and Inspecting 
 
The investigator will permit study-related monitoring, audits, and 
inspections by the Penn State quality assurance program office(s), IRB, 

14 
 
the sponsor, and government regulatory bodies, of all study related 
documents (e.g. source documents, regulatory documents, data collection 
instruments, study data). The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory). 
 
 
17.0 References 
 
[1] Smethers, A. D., Roe, L. S., Sanchez, C. E., Zuraikat, F. M., Keller, K. L., Kling, S. 
M., & Rolls, B. J. (2019). Portion size has sustained effects over 5 days in preschool 
children: A randomized trial. The American Journal of Clinical Nutrition, 109(5), 1361-
1372. 
 
[2] Smethers, A. D., Roe, L. S., Sanchez, C. E., Zuraikat, F. M., Keller, K. L., & Rolls, B. 
J. (2019). Both increases and decreases in energy density lead to sustained changes in 
preschool children's energy intake over 5 days. Physiology & Behavior, 204, 210-218. 
 
[3] USDA Food and Nutrition Service. Child and Adult Care Food Program (CACFP). 
Nutrition Standards for CACFP Meals and Snacks. Version current: 26 May 2020 
[Internet]. Available from: https://www.fns.usda.gov/cacfp/meals-and-snacks. 
 
[4] Littell R.C., Milliken G.A., Stroup W.W., Wolfinger R.D., Schabenberger O. SAS for 
Mixed Models, 2nd ed. SAS Institute Inc., Cary, NC, 2006.