Longitudinal changes in nutrient levels of human milk have been observed with the progression of lactation, but less is known about changes at different feedings during a 24-h period. In this study, calcium, magnesium, and zinc levels were examined in milk obtained from 49 women at every breast feeding during one 24-h period each month for the first 6 months of lactation. Magnesium and zinc concentrations showed significant diurnal variation at each stage of lactation examined, whereas calcium levels were relatively constant. Maximum variations of 7.5, 17, and 48% were observed within a 24-h period in mean levels of calcium, magnesium, and zinc, respectively. Irrespective of variability, zinc and magnesium concentrations in a single milk sample obtained between 10:00 a.m. and 2:00 p.m. were highly correlated with the mean value of samples collected at each feeding during a 24-h period.