OBJECTIVE: The aim of this study was to test associations of prepregnancy BMI, gestational weight gain, oral glucose challenge test results, and postpartum weight loss as predictors of breast milk leptin, insulin, and adiponectin concentrations and whether these relationships vary over time. METHODS: Milk was collected at 1 and 3 months from 135 exclusively breastfeeding women from the longitudinal Mothers and Infants Linked for Healthy Growth (MILk) study. Hormones were assayed in skimmed samples using ELISA. Mixed-effects linear regression models were employed to assess main effects and effect-by-time interactions on hormone concentrations. RESULTS: In adjusted models, BMI was positively associated with milk leptin (P < 0.001) and insulin (P = 0.03) and negatively associated with milk adiponectin (P = 0.02); however, the association was stronger with insulin and weaker with adiponectin at 3 months than at 1 month (time interaction P = 0.017 for insulin and P = 0.045 for adiponectin). Gestational weight gain was positively associated and postpartum weight loss was negatively associated with milk leptin (both P < 0.001), independent of BMI. Oral glucose challenge test results were not associated with these milk hormone concentrations. CONCLUSIONS: Maternal weight status before, during, and after pregnancy contributes to interindividual variation in human milk composition. Continuing work will assess the role of these and other milk bioactive factors in altering infant metabolic outcomes.