Study protocol
This was a three-way repeated measurement randomized, controlled cross-over trial in which each individual served as his/her own control. The study protocol was approved by the ethics committee of the School of Public Health, Shahid Sadoughi University of Medical Sciences (Identification number: IR.SSU.SPH.REC.1394.104) and was registered on 9th October 2016 in the Iranian registry of clinical trials (IRCT, www.irct.ir, registration ID: IRCT2016072012571N5; URL: http://en.irct.ir/trial/12621). The purpose of the study was explained to all parents of the children participating in the study and to their school authorities (principles, assistants, and teachers), and written consents were obtained from the parents.
Randomization and blinding
The present study tried to examine the effect of three intervention drinks [skim milk:SM (0% fat), whole milk:WM [3% fat], and apple juice:AJ] on satiety and short-term energy intake in the context of a three-way randomized cross-over trial. Therefore, participants were randomly assigned into 6 rolling methods (SM-WM-AJ, SM-AJ-WM, WM-SM-AJ, WM-AJ-SM, AJ-WM-SM, and AJ-SM-WM) to receive the three iso-volumic drinks along with a fixed energy breakfast (Fig. 1). The randomization was done using statistical package for social sciences (SPSS) software. Intervention periods lasted for 2 consecutive days, and there were 5 days of wash-out period between the interventions. We selected two consecutive days because our previous experience showed that two days of intervention is enough to show the effect of low-fat milk intake on satiety and short-term energy intake [7]. The whole and the skim milk were coded by a researcher out of the study and provided for the study personnel. Therefore the personnel and the participants were blinded to the type of milks. However it was not possible for the researchers to blind the participants to the apple juice.
Eligibility criteria
Participants were selected from two elementary schools (one for girls and one for boys) Isfahan, Iran. Children aged 10–12 years old who were with obesity based on the reference curves provided by WHO (obesity was defined as having a body mass index (BMI) above the 95th percentile based on WHO BMI-for-age charts), were not on a special diet or had no intention to go on diet during the study period, did not have intolerance to cow’s milk, did not report any history of congenital or metabolic diseases using information provided from parents, and regularly consume breakfast were included in the present study. It was planned to exclude the participants if they denied participating in at least two of the intervention periods.
Intervention details
On the intervention days, boys and girls attended school, while they were in the fasted state at 7 a.m. Subjects were given breakfast meal that was composed of 65 g of an Iranian whole-wheat bread (called sangak), 15 g of low-fat cheese and 12 g of walnut with one of the three test beverages: skim milk, apple juice or whole milk (full-fat) milk. The food and nutrient content of the breakfast along with the test beverages are provided in Additional file 1: Table S1. Subjects were checked to completely eat their breakfast till 7:30 a.m. After breakfast, the study attendants were prohibited to eat and drink anything except water and a small portion of fruit (apple) that contained approximately 15 g of carbohydrates, until lunchtime. A lunch meal was provided at 12:30 for all of the participants in the schools’ hall. The food items provided for lunch were the same for all participants and intervention periods. The food items which were provided for the first day of each intervention period were cooked rice, kebab, yogurt, cola beverage, pasta and bottled water and the food items provided for the second intervention days were cooked rice, chicken, yogurt, bottled water and cola beverage. Subjects were allowed to freely select their food. Participants were asked to continue to eat until they felt absolutely full. The food items were provided in packages and each food item was weighed and then included in its package. Participants were allowed to select as much as packages they want. After the participants finishing their lunch, the remaining foods in each package were weighted.
Anthropometric measurements
A wall-mounted stadiometer was used to measure the height, and the weight was assessed using a digital scale, whereas subjects were wearing minimal clothing. BMI was computed as weight (kg) divided by the square of height (m2). Anthropometric measurements were performed three times for each person and the value that was shown at least twice for each subject was recorded.
Assessment of the appetite
The participants filled a visual analogue scale (VAS) which was previously used in children [13] before consumption of the breakfast (T0), right after breakfast consumption (T1) and each hour after breakfast until before lunch [8:30 a.m. (T2), 9:30 a.m. (T3), 10:30 a.m. (T4), 11:30 a.m. (T5), 12:30 a.m. (T6)]. Participants were trained by the study members on how to complete the questionnaire. Each VAS consisted of 4 questions: “How hungry do you feel at this moment?”, “How full do you feel at this moment?”, “How strong is your desire to eat at this moment?” and “How much food do you think you could eat at this moment?” which assessed the components of appetite including the desire to eat, fullness, hunger, and prospective consumption respectively. The answers to these questions were made through placing a cross on the 100-mm lines anchored on their end with the most positive and the most negative answers for each question (I do not feel hungry at all/I feel very hungry, I do not feel satiety at all/I feel very satiety, I have no desire/I so desire, no/very much). The scores for each of these questions would be from 0 to 100; therefore, the total score for appetite ranged from 0 to 400.
Assessment of the energy intake and the physical activity
Subjects were asked to continue their habitual diet and physical activity during the study period. The dietary intakes consumed by participants on intervention days from breakfast till lunch was measured by weighted dietary record done by an educated nutritionist. Parents were also asked to complete food records for 2 days before, during and 2 days after the intervention days. We converted the food items to grams using household measures and then converted them to energy using nutritionist IV software (version 3.5.2, Axxya Systems, Redmond, Washington, USA). Parents were also asked to record their children’s physical activity 2 days before the intervention, during the intervention days and 2 days after the intervention using predefined forms. All activities and their duration were recorded by parents during the mentioned periods. The recorded activities were converted to metabolic equivalent-hour/day (Met-h/day) using the MET intensity of each activity [14]. We tried to compare the energy intake and physical activity before the three intervention periods because the physical activity and the energy intake before the intervention days may affect the satiety and the energy intake on the intervention days [15, 16].
Statistical analysis
Descriptive statistics are presented as means ± Standard deviations (SDs) or standard error of means (SEs) where provided. Normality of the outcome variables was assessed using Kolmogorov-Smirnov test. Differences in 1) mean energy intakes at lunch and 2) mean appetite scores, were compared using generalized linear model (GLM) repeated measures procedure considering the test beverages and testing occasions as the repeated factors. Participants’ rolling method, age, and BMI were controlled in different models as between-subject variables. Gender was also considered as a between subjects factor in the analyses for total population. All analyses were done for the total population as well as either gender. P values less than 0.05 (2-tailed) were considered as statistically significant. The randomization and the statistical analyses were conducted by the use of statistical package for social sciences software version 20 (IBM SPSS, Tokyo, Japan).