Background: Daily variations of macro- and trace-elements in human milk (HM) are not well characterised and sampling protocols are highly variable between studies. Objectives: To investigate diurnal and within-feed variation of HM macro- and trace-elements using pre- and post- feed concentrations and to compare infant intake estimates using limited samples with measured 24-hour intake. Methods: HM Samples were collected pre- and post- every feed in a 24-hour period from 11 mother-infant dyads. Test-weighing was used to determine the volume of HM consumed in each feed. For macro- and trace-elements within-feed and daily variation was measured. Intake estimated from a morning pre-feed sample was compared to the measured milk intake calculated from every feed over 24-hours. Macro- and trace-elements concentrations were measured using ICP-MS. Linear mixed modelling was used for statistical analysis. Results: Average intake of HM was 737 ± 63 mL for infants aged 1–6 months and 508 ± 50 for infants aged 6–12 months. Pre- and post-feed HM variation was found for phosphorus, calcium, manganese, iron, copper, zinc, selenium, molybdenum, and iodine (p < 0.05). Variation across 24 h was found for magnesium, phosphorus, potassium, manganese, iron, and selenium (p < 0.05). Estimated intake using morning, pre-feed samples resulted in significantly lower intake when compared to measured milk intake for iron, phosphorus, selenium, and manganese (p < 0.05). Conclusion: Standardised sampling protocols using large sample volumes and multiple collections over 24-hours provide a calculated intake that is more reflective of actual infant HM macro- and trace-elements intake.