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Life sciences · Journal article
Frontiers in Nutrition · October 22, 2026
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Objective In the Saliva Insulin as Biomarker (SIB) randomized controlled trial (RCT), saliva insulin increased more, within 60 min, after one serving of apple juice, and normalized less, after no drink, compared to the acute change after one serving of drinking water. This paper tested for corresponding drink treatment effects on metabolomic profile, recognizing that effects on insulin, a metabolic switch, imply co-occurring effects on many metabolic pathways, simultaneously, and potential for school-based drinking water interventions to efficiently target risk factor profiles. Metabolic pathways potentially impacted by each drink treatment were described by baseline weight condition to generate hypotheses about potential metabolic targets for drinking water intervention. Methods Untargeted metabolomic assays were completed for saliva from 105 healthy children (5–10y), who participated in the 3-arm parallel group SIB RCT (NCT05211843), prior to a routine dental check-up on a weekday morning or afternoon. Unstimulated saliva was collected, at baseline, before study participants were randomly assigned to drink 500 mL water (W), 200 mL apple juice (AJ), or no drink (ND), and 45–60 min post-drink treatment. Randomization and analysis were stratified by age-and-sex-specific BMI percentile (5–85th or >85th). Drink treatment effects on change in saliva metabolomic profiles were compared using orthogonal partial least squares discriminant analysis (OPLS-DA), permutational multivariate analysis of variance (PERMANOVA), and Metaboanalyst 6.0 pathway module. Results W significantly altered the saliva metabolomic profile compared with AJ in children with normal weight (validated OPLS-DA, Q 2 = 0.30, permutation p = 0.005; PERMANOVA p = 0.04) and compared with ND in children with overweight or obesity (Q2 = 0.29; permutation p = 0.04; PERMANOVA p = 0.02). Increases or decreases of 1.5-fold or more in over 100 metabolites with KEGG IDs, after W instead of either AJ or ND, potentially impacted dozens of metabolic pathways. Conclusion In healthy children, with normal weight, overweight or obesity, W instead of AJ or ND caused acute changes in saliva metabolomic profile that signal potential for multiple weight- and drink treatment-specific metabolic targets for drinking water intervention. The results warrant further research to pursue potential metabolic benefits of drinking water. Clinical trial registration NCT05211843, clinicaltrials.gov