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The daily variation in human milk (HM) hormones and macronutrients is not well characterised and sample protocols are highly variable between studies. During a 24 h period, the mothers (n = 10) hand-expressed small milk samples, immediately before and after each breastfeeding or expression from each breast. Test-weighing was used to determine the volume of HM consumed in each feed. Concentrations of leptin, adiponectin, insulin, fat, and glucose were measured by variable biochemical assays. A linear mixed model was fitted to eleven outcomes of leptin, adiponectin, insulin, fat, glucose (dose and concentration for each), and feed volume. The explanatory variables considered were a circadian cycle (cosine and sine terms), and for dose and concentration outcomes, an indicator variable for pre- or post-feed. The random effect was a circadian cycle (cosine and sine terms) for each mother.