Effect of Energy Density Over 5 Days in Preschool Children
Phase: NA
Conditions: Feeding Behavior
Sponsor: Penn State University

 
 
 
 
Effect of Energy Density Over 5 Days 
in Preschool Children 
 
 
ClinicalTrials.gov Protocol ID: ChildFood502 
ClinicalTrials.gov ID Number: NCT03010501 
 
 
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: Study00001824 
Initial Approval Date: 28 January 2015 
IRB Protocol Document version 0.13 
Approval Date: 11 July 2018 
 
 
 
 
 

 
 
Table of Contents 
 
  1.0 Objectives 
  1 
  2.0 Background 
  1 
  3.0 Inclusion and Exclusion Criteria 
  2 
  4.0 Recruitment Methods 
  2 
  5.0 Consent Process and Documentation 
  3 
  6.0 Study Design and Procedures 
  4 
  7.0 Data and Specimen Banking For Future Undetermined Research 
  6 
  8.0 Statistical Plan 
  6 
  9.0 Confidentiality, Privacy and Data Management 
  8 
10.0 Data and Safety Monitoring Plan 
  9 
11.0 Risks  
  9 
12.0 Potential Benefits to Subjects and Others 
  9 
13.0 Sharing Results with Subjects 
  9 
14.0 Economic Burden to Subjects 
  9 
15.0 Other Approvals 
10 
16.0 Adverse Event Reporting 
10 
17.0 Study Monitoring, Auditing and Inspecting 
10 
18.0 References 
11 
 
 

1 
 
1.0 Objectives 
 
1.1 Study Objectives 
Aim: To determine the effect of varying the energy density of foods served over five 
days on energy intake in preschool children. 
 
Hypotheses: 
1. Mean daily energy intake will be greater when children are served higher-energy-
dense versions of foods over five days than when they are served lower-energy-dense 
versions of the same foods over five days. 
2. Daily energy intake in the conditions with higher and lower energy density will begin 
to converge across the 5-day period. 
3. Energy intake across the 5-day period will be adjusted closer to baseline in response 
to decreases in energy intake than in response to increases in energy intake. 
 
1.2 Primary Study Endpoints 
1. Daily intake of food and beverages by energy (kilocalories) 
 
1.3 Secondary Study Endpoints 
2. Daily intake of food and beverages by weight (grams) 
3. Daily intake of food and beverages by energy density (kilocalories/gram) 
 
 
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 [1-4], we have found that increases in both 
food portion size and energy density have been robust in promoting excess energy 
intake in preschool children over one to two days. 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, 
estimated to be three to four days, to determine whether children adjust energy intake in 
response to differences in energy density in the eating environment. 
 
2.2 Previous Data 
See references 1 through 4 
 
2.3 Study Rationale 
This study will allow us to investigate preschool children’s ability to regulate their energy 
intake and to assess how energy density affects intake over a longer period of time. 
 
 

2 
 
3.0 Inclusion and Exclusion Criteria 
 
3.1 Inclusion Criteria 
1. Children enrolled in one of the participating child care centers 
2. Children who are between the ages of 3 and 5 years old at the time of enrollment 
3. Parents (18 years or older) of children enrolled in the study 
4. Teachers (18 years or older) in a classroom with enrolled children 
 
3.2 Exclusion Criteria 
1. Children with allergies or restrictions to any of the foods served 
2. Children with health issues that preclude participation 
3. Children who are unavailable for the entire duration of the study 
 
3.3 Early Withdrawal of Subjects 
 
3.3.1 Criteria for removal from study 
There are no criteria for removal of subjects from the study. All participation is 
voluntary and a child can refuse a meal 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 
Any child and parent in a participating classroom of the child care center may 
participate. Teachers of participating children in the classroom will also be asked to 
participate. 
 
4.2 Recruitment process 
We will recruit through a parent letter placed in each child's mailbox. A study staff 
member who is approved (in this application) to do recruitment and consenting of 
subjects will be available in the center during child pick-up to answer any questions 
parents may have. 
Teachers in classrooms will be verbally asked to participate 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 verbally asked to participate 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 or restrictions to the foods served in the study will not be eligible 
to participate. Screening questions regarding food allergies and intolerances will be 
included with the consent form. 

3 
 
5.0 Consent Process and Documentation 
 
5.1 Consent Process 
 
 
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 child care 
center by a trained member of the study staff. 
Teachers will consent to fill out a questionnaire about enrolled children prior to 
the first study week in their classroom at the child care center. 
An addendum consent was added to this study due to university closure for 
inclement weather. Since five consecutive days are needed for data collection, 
Study Week 2 was restarted for one cohort. 
Only parents will be given the consent addendum as teacher participation does 
not change with this rescheduling. Parents will be given the addendum at the 
child care center. Consent will be obtained at the child care center by a trained 
member of the study staff. 
5.1.1.2 Coercion or Undue Influence during Consent 
Parents will be given the 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 form 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. Participation in the changed schedule is 
voluntary. 
A parent of the participant will sign the consent form. Parents will be given a copy of 
the consent form to keep for records. 
A parent of the participant will sign the consent addendum. Parents will be given a 
copy of the consent addendum to keep for their 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 
N/A 
 
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 
5.3.2.2 Adults Unable To Consent 
N/A 

4 
 
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. 
5.3.3.2 Assent 
Children can refuse to eat any meal if they choose. Before height and weight are 
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 crossover study has a within-subjects design. There will be three experimental 
conditions; one condition in which meals of lower energy density (ED) are served, one 
condition in which meals of baseline energy density are served, and one condition in 
which meals of higher energy density are served. The order of conditions across the 
three 5-day periods will be counterbalanced across classrooms. Children will be served 
the same amount (weight) of food and beverages across all three conditions. In the 
lower-ED condition, the amount of energy served over each day is ~225 calories less 
than in the baseline condition. Energy served over the day in the higher-ED condition is 
~225 calories more than in the baseline condition. Daily menus in all conditions meet 
the minimum guidelines for energy and food groups outlined by the USDA and the Child 
and Adult Care Food Program (CACFP). 
 
6.2 Study Procedures 
1. Over 5 consecutive days, enrolled children will be served breakfast, lunch, dinner, 
and snacks of foods commonly served in the child care center according to their regular 
meal and snack schedules. Study menus were developed from the menus at each child 
care center to be similar to the foods that children are regularly served. Milk or water will 
be served as beverages. Meals will be served at tables of 3 to 6 children and one adult, 
which is a standard practice at the child care 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 
experimental conditions, which differ only in food energy density. The lower-energy-
density condition will provide foods 20% lower in energy density than the baseline 
condition, and the higher-energy-density condition will provide foods 20% higher in 
energy density than the baseline condition. Each participant will be served individual 
pre-weighed meals. All food and beverage items will be weighed with a food scale 
before and after meals to within 0.1 grams 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 study staff 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 
child care center the following day to be weighed. 

5 
 
2. 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 there is any deviation from this 
procedure, parents will be asked to report the extra food and beverage consumption. 
3. Physical activity levels will be measured using tri-axial accelerometers worn over the 
five study days in each period. The accelerometers will be worn while the children are 
present at the child care center (from before breakfast to after dinner). Study 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. 
4. On the final (16th) visit to the child care center, children will have their height and 
weight measured by a trained member of the study staff. Body weight will be measured 
in triplicate using a portable digital scale, and height will be measured in triplicate using 
a portable stadiometer. The children will also complete the Tasting Game, in which they 
are asked to taste a small food sample and point to a cartoon face that represents their 
liking of the food on a scale of “yucky” to “yummy”. 
5. Parents will be asked to complete an Evening Report form, Background 
Questionnaire, Children’s Eating Behavior Questionnaire, Caregiver’s Feeding Style 
Questionnaire, and Child Feeding Questionnaire. The Evening Report form will be 
completed at the end of each study day and returned each morning to study staff. The 
Background Questionnaire will be completed during the first 5-day study period and 
returned to study staff. The Children’s Eating Behavior Questionnaire, Caregiver’s 
Feeding Style Questionnaire, and Child Feeding Questionnaire will be administered 
during the last 5-day study period. All questionnaires will be completed using paper 
copies. 
6. Photographs may be taken of children eating meals, participating in the Tasting 
Game, or having their height and weight measured. 
7. There will be a 1-week break between 5-day study periods. There will be a 4- to 6-
week wash-out period between this experiment and Experiments 1 and 3 at each child 
care center. We will rotate the participating child care centers to ensure that each child 
completes the wash-out and is eligible to participate in the next experiment if they 
choose. 
8. Approved study staff (on this application) will email parents on the Friday before a 
study week begins to remind parents of the upcoming study week. 
9. Teachers will be asked to complete one questionnaire (Caregiver’s Feeding Style 
Questionnaire) per participating child in their classroom. Teachers will only complete 
questionnaires about children who are enrolled in the study. This questionnaire will be 
administered using a paper copy. Teachers will be verbally informed by study staff 
about the children for whom they are to complete questionnaires. 
10. 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 each study period will be five consecutive days, and each participant will 
be asked to complete three of these 5-day periods. During study days, each meal and 
snack will take approximately 30 minutes to complete. The estimated time required by 
the participant each day is 2.5 hour for meals. The estimated total time required for all of 
the meals is 37.5 hours across 15 days. The height and weight measurements and the 

6 
 
Tasting Game on the final visit will require an additional hour of time. For the parents, 
30 minutes will be required to complete the questionnaires. The total time commitment 
for the study is 39 hours. 
Teachers will spend approximately 20 minutes per questionnaire. 
 
 
7.0 Data and Specimen Banking For Future Undetermined Research 
 
7.1 Data and/or specimens being stored 
N/A 
 
7.2 Location of storage 
N/A 
 
7.3 Duration of storage 
N/A 
 
7.4 Access to data and/or specimens 
N/A 
 
7.5 Procedures to release data or specimens 
N/A 
 
7.6 Process for returning results 
N/A 
 
 
8.0 Statistical Plan 
 
8.1 Sample size determination 
The sample size for the study was estimated from a linear mixed model and an 
approximation technique [5] using exemplary data sets based on prior 1- and 2-day 
studies in preschool children [2,3]. We assessed the required sample size for several 
scenarios with different assumptions about changes in intake on days 3 to 5 that 
represented quadratic trajectories over time. To detect a clinically significant change of 
50 kcal/d over time (movement toward adjustment after 3 to 5 days) with a Type 1 error 
rate of 5% and power of 90%, it was determined that 45 participants would be required. 
To allow for withdrawals and non-compliance to the protocol, at least 55 children will be 
enrolled.  
 
8.2 Statistical methods 
The main outcomes of the study are daily consumption (by weight, ED, and energy) of 
manipulated foods, non-manipulated foods and milk, and their combined total. Other 
outcomes are cumulative intake for the entire 5 days, daily macronutrient intake, daily 
intake of non-manipulated items by meal component (fruits and vegetables at main 
meals, grain-based snacks, dairy snacks, fruit and vegetable snacks, and milk), and 

7 
 
physical activity time (daily step counts, sedentary time, and moderate-to-vigorous 
time). To analyze differences in mean daily outcomes across the 3 experimental 
conditions, linear mixed models with repeated measures are used. The fixed factors in 
the models are ED condition (lower-ED, baseline-ED, or higher-ED), study week, study 
day, menu, classroom, and sex. Interactions between these factors are tested and 
removed from the model if not significant. Participants are treated as a random effect. 
The Tukey-Kramer method is used to adjust for multiple pairwise comparisons between 
means. In order to evaluate the contribution to children's intakes of non-study foods 
reported by parents, the energy content of the reported items is estimated by study 
staff. The outcome of daily energy intake is analyzed both with and without the non-
study items. This estimated data is not included in further analyses, because these 
estimates are based on parental recall and were less accurate than measured intake.  
 
Random coefficients models are used to test whether the trajectories of daily intake in 
the 3 conditions converge over the 5 days, i.e., whether regulation of energy intake 
occurs over time in response to ED variations. The trajectories of intake over time are 
modeled separately for each child and allowed to vary randomly. Time (study day) is 
treated as a continuous covariate in the model and polynomial factors of time (linear 
and quadratic coefficients) are tested as both fixed and random effects to determine the 
shape of the intake trajectory. The intercept of the trajectory reflects the magnitude of 
daily intake. The linear coefficient represents the rate of change in intake (slope) across 
time after Day 1, and the quadratic coefficient represents the rate of acceleration or 
deceleration in intake (curvature) across time after Day 1.  
 
The influence of continuous participant characteristics (age, body weight, height, BMI-
for-age percentile, BMI z-score, step counts, and questionnaire subscales) on the 
relationship between the experimental factors and intake is assessed by analysis of 
covariance with linear mixed models. Because multiple participant characteristics are 
tested as covariates, the method of Benjamini and Hochberg [6] is used to adjust the 
significance levels, with the false discovery rate set at 0.05 [6]. In order to standardize 
for energy needs, models having significant covariance with weight status are adjusted 
for estimated energy requirements [7]. Differences in subject characteristics between 
boys and girls, as well as differences in intake between children with and without 
obesity (sex-specific BMI-for-age percentile ≥85 and<85, respectively), are assessed by 
independent-samples t-tests. Within conditions, the means of daily energy intakes 
expressed as a percentage of estimated energy requirements are compared to 100% 
using a one-sample t-test and controlling the family-wise error rate using the Bonferroni 
correction.  
 
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. To evaluate whether 
the distribution of taste ratings (yummy, just okay, yucky) of the lunch and snack dishes 
differs significantly across experimental conditions, ordinal logistic regression is used; 
results are reported as odds ratios (OR) with 95% confidence intervals. Outcomes from 

8 
 
statistical models are reported as mean ± SEM and subject characteristics are reported 
as mean ± SD. Standardized effect sizes are calculated using Cohen's d statistic, with 
no adjustment for correlation due to repeated measures. Results are considered 
significant at P < .05. All data are analyzed using SAS software (SAS 9.4, SAS Institute, 
Inc., Cary, North Carolina, USA).  
 
 
9.0 Confidentiality, Privacy and Data Management 
 
9.1 Confidentiality 
We may take photographs of children eating meals, participating in the Tasting Game, 
or having their height and weight measured, which may be used for poster or oral 
presentations at scientific meetings. Parents will indicate on their consent form if they 
agree to have their child’s photograph taken. All images will be stored on a password-
protected network drive which can be accessed only by those identified on this 
application. Images will be stored with a generic title and will not be labeled with any 
identifiable information. The images will be deleted from the network drive within 3 years 
of completion of the study. 
9.1.1 Identifiers associated with data and/or specimens 
Children’s data will only be identified by a 3 digit number, dot color, and a letter of 
the alphabet. Teachers will not have their own identifiers. 
9.1.1.1 Use of Codes, Master List 
The list that links the ID code to subject identifiers will be stored in a locked closet in 
the Lab Manager's office in 226 Henderson Building. Only those listed on this 
application will have access to it. The master list of the codes will be destroyed upon 
publication of the study or five years after the close of the study, whichever comes 
first. 
9.1.2 Storage of Data and/or Specimens 
Paper data with identifiers 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. 
Access to network drives is protected by passwords. 
Paper and digital copies of data with identifiers removed will be stored indefinitely. 
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. 
9.1.3 Access to Data and/or Specimens 
Only those listed on this application will have access to data. 
9.1.4 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. 
 
 
 

9 
 
9.2 Privacy 
No personal information other than first name, telephone number, and email address is 
stored where it can be seen by study staff. Participant data will remain unidentified 
except by study number and letter. Only senior staff members listed on this application 
have access to personal information such as address, age, weight, etc. 
Data on food consumption is collected in a separate room where only study staff is 
present. Heights and weights for both children and parents are collected in a private 
area of the child’s classroom. 
 
 
10.0 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 child care centers. 
 
 
11.0 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 child care center and at home, which include the risk of food allergy, food-
borne illness, and choking on food. 
The amounts served in all conditions meet or exceed minimum requirements for calorie 
and nutrient content for this age group as specified by the UDSA and the Child and 
Adult Care Food Program (CACFP). 
There are no potential risks for teacher participants. 
 
 
12.0 Potential Benefits to Subjects and Others 
 
12.1 Potential Benefits to Subjects 
There are no direct initial benefits to subjects participating in the proposed study. 
 
12.2 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. 
 
 
13.0 Sharing Results with Subjects 
No results will be shared with the subjects. 
 
 
14.0 Economic Burden to Subjects 
 
14.1 Costs 
N/A 
 
14.2 Compensation for research-related injury 

10 
 
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. 
 
 
15.0 Other Approvals 
 
The Bennett Family Center, The Child Care Center at Hort Woods, and Step by Step 
School for Early Learning have given permission to conduct this study in their facilities. 
 
 
16.0 Adverse Event Reporting 
 
16.1 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 reported to the IRB in accordance 
with the IRB policies and procedures. 
 
16.2 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 etc.). The investigator will ensure 
the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
 
17.0 Study Monitoring, Auditing and Inspecting 
 
17.1 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 etc.). The investigator will ensure 
the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
 
 
 

11 
 
18.0 References 
 
[1] K.E. Leahy, L.L. Birch, B.J. Rolls. Reducing the energy density of an entrée 
decreases children's energy intake at lunch. J. Am. Diet. Assoc. 108 (2008) 41–48. 
 
[2] K.E. Leahy, L.L. Birch, B.J. Rolls. Reducing the energy density of multiple meals 
decreases the energy intake of preschool-age children. Am. J. Clin. Nutr. 88 (2008) 
1459–1468. 
 
[3] M.K. Spill, L.L. Birch, L.S. Roe, B.J. Rolls. Hiding vegetables to reduce energy 
density: an effective strategy to increase children's vegetable intake and reduce energy 
density. Am. J. Clin. Nutr. 94 (2011) 735–741.  
 
[4] S.M.R. Kling, L.S. Roe, K.L. Keller, B.J. Rolls. Double trouble: portion size and 
energy density combine to increase preschool children's lunch intake. Physiol. Behav. 
162 (2016) 18–26. 
 
[5] R.C. Littell, G.A. Milliken, W.W. Stroup, R.D. Wolfinger, O. Schabenberger. SAS for 
Mixed Models, 2nd ed. SAS Institute Inc., Cary, NC, 2006. 
 
[6] Y. Benjamini, Y. Hochberg. Controlling for the false discovery rate: a practical and 
powerful approach to multiple testing. J. R. Stat. Soc. Ser. B, Stat. Methodol. 57 
(1995) 289–300. 
 
[7] N.F. Butte, W.W. Wong, T.A. Wilson, A.L. Adolph, M.R. Puyau, I.F. Zakeri. Revision 
of dietary reference intakes for energy in preschool-age children. Am. J. Clin. Nutr. 
100 (2014) 161–167.