The study was approved by the Ethics committee of the Institute for Medical Research, University of Belgrade (Dossier No. EO123/2017), and all procedures involving human subjects were in accordance with the Declaration of Helsinki. Written informed consent was obtained from each participant. In the case of children younger than 18 years of age, a parent gave consent and the child gave assent. The interviews were conducted between July and December of 2017.
The study sample was designed to be representative of the Serbian population as determined by the latest census from 2011 . Sampling was stratified by age groups (10–17, 18–64, 65–74), by gender, and by region of residence. The regions of residence in Serbia were Vojvodina, South East Serbia, West Serbia and Belgrade. Recruitment was organized and conducted by project team members at the household level, with not more than one individual recruited per household. The survey plan was structured to capture a representative proportion of weekdays and weekend days for the study sample. Interviewers were advised to organize home visits to participants whenever possible. If it was not possible, another interview site was agreed upon depending on the region and location of the interviewer’s office. For the present analysis, pregnant women (n = 15) were excluded, resulting in a total of 334 non-pregnant subjects.
Two interviews were done with each subject, at least 1 week apart. The first interview consisted of collecting anthropometric data, administering a demographic questionnaire, and a 24-h dietary recall (see section on dietary assessment below). All of the initial interviews were done in person. Anthropometric data (height, weight) were measured for 65% of the study population. For the rest, self-reported values were collected. The study questionnaire collected demographic information on age, gender, marital status, place of residence, ethnicity, employment, education, smoking, height, weight, physical activity and health status.
Physical activity was assessed using the International Physical Activity Questionnaire short form that has been published . This included questions on how much time was spent on moderate, vigorous and walking activities in the past 7 days (number of days and hours or minutes each time). For walking, only bouts of at least 10 min were enumerated. Time spent sitting was captured using questions that asked about time spent sitting on weekdays and on weekends. If a participant was not sure of time spent on physical activity, a time of 10 min/day was input, and this was needed for 23 of the questionnaires. The sitting and physical activity data was used to derive hours per week of activity or of sitting.
The dietary assessment was conducted following the strict guidelines of the European Union (EU) Menu methodology as published previously . In order to estimate usual dietary intakes, two non-consecutive 24-h recalls of diet were conducted with an interval of at least a week between the two recalls . A multi-pass method was used to maximize accuracy of the recalls . The multi-pass dietary recall utilized a visual for estimating serving sizes: a pictorial food atlas that was developed for the Balkan region to facilitate precise estimation of portion sizes as published by our group . The first recall was always performed as a face-to-face interview and the second recall was conducted face-to-face or via phone, based on participant’s availability and preference.
Daily food and nutrient intakes were calculated using the Diet Assess & Plan software (DELTA Electronic Ltd., Subotica, Serbia). This is an advanced dietary assessment and nutrition planning software tool that has been used previously in national, regional and international nutritional surveys, and it was evaluated in the European Food Safety Authority (EFSA) ring trial involving six countries [18,19,20]. Nutrient intake calculations were performed using the Serbian Food Composition Database . As published previously, the database was harmonized with European Food Information Resource Network of Excellence (EuroFIR) standards and embedded in the EuroFIR platform and represents the core element of the Balkan Food Platform . Average daily intakes for each subject were derived as a mean of the two 24-h recalls.
The time of each eating occasion reported during the 24-h dietary recall was recorded. The time noted represents the start of the eating occasion. This varied across the 2 days of intake, and the times for each eating occasion for each subject across the 2 days of intake were averaged. The eating occasions were denoted in the data output as before breakfast, breakfast, morning snack, lunch, afternoon snack, dinner, and after dinner snack. For some subjects, there were two afternoon snacks, resulting in a maximum of eight eating occasions that were recorded for each subject. This data was used to calculate average calories consumed by each subject before 16:00.
Five age categories were created to evaluate differences in diet and BMI by age. The first category was for children ages 10–17. Adults ages 18–64 were categorized into tertiles. Since some people were of the same age, the tertiles are not precisely equal in number. The last category was for adults ages 65–74 years since the retirement age in Serbia is 65 years.
Diet Quality Score (DQS)
We computed a DQS for adults to evaluate how many dietary recommendations were being met by each adult subject. This analysis focused on adults only since the number of children in the sample was smaller, dietary recommendations in children differ from that in adults, and health issues are different in children than in adults. The DQS summed the number of dietary recommendations met by each subject.
The dietary recommendations vary from country to country and change over time. We focused on those dietary factors for which healthy eating recommendations have been established as summarized by the European Union (EU) Science Hub of the European Commission . The DQS therefore included one point for meeting each of the EU Science Hub recommendations for fruits and vegetables, fiber, saturated fat, sugar, and sodium as detailed below, resulting in a score of 0–5.
For fruit and vegetable intakes, recommendations have ranged from 5 servings a day to more than 13 per day, given in ½ cup servings [21, 22]. The World Health Organization in 2006 recommended a daily intake of fruit and vegetables of at least 400 g/day . Using an estimate of 85 g vegetables or fruit in a ½ cup serving, 400 g/day is about 5 servings/day. Intakes of 400 g/day or higher therefore received one point. Fiber recommendations are typically 25–35 g/day, often provided in a sex-specific and age-specific manner. An adequate intake for both children and adults is 14 g/1000 kcal in the Dietary Guidelines for Americans, which harmonizes well with the European recommendations . We therefore awarded one point for meeting or exceeding an intake of 14 g fiber/1000 kcal.
Recommendation for sugar intake generally include consuming less than 10% of energy from added sugars, and we used that recommendation for awarding a point in the DQS . For fat intake, moderate intakes of total fat (20–35% of energy) have been recommended in the past, but more recent evidence shows little associations of total fat intake with disease risks . The recommendations have been more consistent for limiting saturated fat, and we therefore used less than 10% of calories from saturated fat as the cut-off to award a point in the DQS. For sodium intake, recommendations are to limit intake to 2.0–2.4 g/day, and we awarded a point towards the DQS for sodium intakes less than 2.3 g/day .
Whole grain intake was not available from our data set and was not used as a criterion, but this likely does overlap with fiber intake which we did include. Protein and water intakes also were not used in the score. An average requirement of 0.60–0.66 g protein/kg body weight has been set as a recommendation across countries . In our population sample, only 14 subjects (5 children and 9 adults) reported consuming less than 0.6 g/kg protein, and the mean was 1.1 g protein/kg for adults. Protein intake therefore was not used as criterion in the DQS. Recommendations for water intakes have not been widely made and the recommendations vary from 1000 to 2700 ml/day . This made it difficult to select a scoring criterion for water intake and it was omitted from the DQS.
To generate a continuous DQS, we constructed a Diet Quality z-Score. Normalized intakes for each nutrient or food were calculated as the difference between each subjects’ value and the recommended value divided by the standard deviation. The differences were taken using positive values for dietary intakes that exceed the recommendations for fiber (14 g/1000 kcal) and fruit/vegetable intakes (400 g/day), and negative values for dietary intakes that exceeded the recommended limit for saturated fat (10% of energy), sodium (2300 mg/day) and sugar (10% of energy). The Diet Quality z-Score was calculated by summing the five normalized nutrient or food scores.
Data from the demographic questionnaire and from the nutrition analysis program was maintained in Excel. All statistical analyses were conducted in IBM SPSS Statistics for Windows, Version 24.0. (IBM Corp., Armonk, NY). Comparisons between groups (adults versus children, or early versus late eaters), were made using two-sample, two-sided t-tests for continuous variables or by using Pearson Chi-square tests for categorical variables. Correction of p-values for false discovery rates (FDR) was done using the method of Benjamini and Hochberg . Trends of DQS versus percent of calories consumed before 16:00 were explored using linear regression models, adjusting for participant age and gender. Comparisons of energy intakes in three ordinal categories of DQS, defined by DQS scores of 0–1, 2–3, or 4–5, were evaluated using ANOVA, and natural log transformation of the data to achieve normality was used for the kcal/day variable. The Tukey honest significance test was used post-hoc to control for multiple comparisons.
To evaluate the effects of age category and gender on dietary intakes, linear regression models for each of the key dietary intake outcomes were constructed (fruit and vegetables, fiber, saturated fat, sodium and sugar). These models included age category, gender and the interaction between age and gender as predictors. When the interaction was not significant it was removed to assess the main effects of age category and gender.
Calorie content across the three main meals, breakfast, lunch and dinner (Fig. 1), was compared using linear mixed models with kilocalories per meal as the outcome. The model included meal as a categorical covariate and a random intercept was used to account for within person correlation. Separate models for children and adults were constructed, and an overall model with an interaction between age group and meal was also investigated. We also calculated the timing of the largest meal of the day, averaged across 2 days of assessment for each subject, based upon energy content of the reported meals. Histograms were constructed to evaluate the timing of when the largest meal was consumed by adults who and do not work outside the home. Reasons for not working outside the home included being retired, currently unemployed, disabled or working as a homemaker.
Mean energy intakes (with SD) across eating occasions in 334 adults and children. Breakfast was at about 9 am, lunch at 3 pm and dinner at 8 pm. The other eating occasions were reported as snacks. For both children and adults, linear mixed models indicated that lunch had a significantly higher calorie content than either breakfast or dinner, and there were no significant differences in calorie content between breakfast and dinner