2.1. Pilot study design and Population

This study was a subproject of the Harmonized Micronutrient Project (ClinTrials.gov # {"type":"clinical-trial","attrs":{"text":"NCT01823744","term_id":"NCT01823744"}}NCT01823744) that analyzed omics, biochemical, and nutritional status (Mathias et al., ²⁰¹⁸) at baseline (time point or visit 1); after 6 weeks of daily supplementation of vitamins and minerals (time point or visit 2); and after 6 weeks of a washout period (time point or visit 3). Participants were healthy children and adolescents (ages 9–13) recruited from the west side of Ribeirão Preto (Brazil) in two county schools and one private school. For this specific pilot study, 20 participants were randomly selected from a sample size of 136 children and adolescents that were previously included following specific exclusion criteria: (a) one or more episodes of axillary temperature higher than 37°C within the 15 days preceding the data collection, (b) three or more episodes of liquid stools within the 24 hr before assessment, (c) current intake of vitamin or mineral supplement; dietary restrictions of any time, including weight‐loss interventions, and (d) history of chronic diseases; participation in another clinical trial within the 4 weeks preceding the study (Mathias et al., ²⁰¹⁸). The 20 participants were divided into two metabolic groups according only to their lipid profiles: individuals in pool 1 (n = 10) had lower triglycerides, LDL, and VLDL levels, and individuals in pool 2 (n = 10) had higher triglycerides, LDL, and VLDL levels at baseline. The participants were evaluated by a pediatrician to determine their clinical conditions and pubertal stage, according to Tanner's criteria (Tanner, ¹⁹⁶²) in visit 1, 2, and 3.

All participants received a daily supplement of 12 vitamins and 5 minerals in a commercial milk bar (Nestrovit^®) (Table ​(Table1)1) for 5 days per week for 6 weeks. This product was chosen because it (a) was palatable (which would facilitate acceptance by participants), (b) had low amounts of calories (3 milk bars contains 75 calories), (c) has been commercially available in Switzerland since 1936 but never sold in Brazil, and (d) had a known and standard nutritional composition, all of which met the objectives of this study. Six of the authors individually monitored supplement intake at the beginning of each school period, and therefore, the compliance rate for the individuals in this substudy was 100%.

Milk Bar Composition by tablets and comparisons with Dietary Reference Intakes (DRIs) and Upper Tolerable Intake Levels (UL)

Comercial name: “Nestrovit”, brand: “Nestlé”. 2 milk bars (10 g): 51.3 kcal, 4.4 g of carbohydrate, 0.5 g of protein and 3.4 g of lipid. 3 milk bars (15 g): 77 kcal, 6.7 g of carbohydrate, 0.8 g of protein and 5.2 g of lipid.