There is an inadequate understanding of the daily variations in hormones and macronutrients in human milk (HM), and sample collection protocols vary considerably from study to study. To investigate changes in these milk components across 24 h, 22 lactating women collected small milk samples before and after each breastfeed or expression from each breast. Test weighing was used to determine the volume of HM consumed in each feed. The concentrations of leptin, adiponectin, insulin, fat, and glucose were measured, and the intakes were calculated. A linear mixed model was fitted to assess within-feed and circadian variation in HM feed volume and concentration, and intakes of several components. The average infant intake of HM was 879 g/24 h. Significantly higher pre-feed concentrations were found for adiponectin and glucose and lower post-feed concentrations were found for insulin and fat. Significant circadian rhythms were displayed for leptin, adiponectin, insulin, glucose (both concentration and intake), fat concentration, and milk volume. These findings demonstrate the necessity for setting up standardised and rigorous sampling procedures that consider both within-feed and circadian variations in HM components to gain a more precise understanding of the impacts of these components on infant health, growth and development.