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BACKGROUND Play-based activities can be a positive intervention to increase participation of overweight children. Metabolomics can reveal elemental shifts in the metabolome, lending to potential mechanistic explanations behind improvements in physiological systems. OBJECTIVE To elucidate dose-response urinary metabolomic signature shifts in overweight preadolescents following four or eight weeks of supervised play-based activity versus a typical summer break control group. We hypothesized that eight weeks of activity would cause the greatest shift in the metabolites. STUDY DESIGN Twenty-two recreationally active preadolescents (12 males, 10 females) were randomly assigned to a four-week (4w) or eight-week (8w) activity group or to a control group (C). Participants reported to the laboratory on two separate occasions during which descriptive characteristics were recorded and urine samples were obtained. Children in the 4w and 8w cohort were tested at the beginning and end of the four and eight weeks of a supervised play-based physical activity program where they were active for 6 hours a day, 5 days a week. Children in the C group were tested before and after eight weeks of an unsupervised summer break. RESULTS A valid supervised partial least squares discriminant analysis model was obtained between post-exercise subjects in 8w and C (3 components, R2X = 0.332, R2Y = 0.976, Q2 = 0.091). The eight week intervention yielded significant metabolomic changes in several identified compounds. CONCLUSION When compared to a typical unsupervised summer break, a supervised play-based intervention provides enough of a stimulus for a shift in the metabolome.