OBJECTIVE To estimate fat and energy contents of human milk during prolonged lactation. METHODS Thirty-four mothers, of term, healthy, growing children, who had been lactating for >1 year (12-39 months) were recruited. Control subjects were 27 mothers, of term infants, who had been lactating for 2 to 6 months. Fat contents of the milk samples were estimated as creamatocrit (CMT) levels. Energy contents of the milk were measured with a bomb calorimeter. RESULTS The groups did not differ in terms of maternal height and diet, infant birth weight, gestational age, or breastfeeding frequency. They differed significantly in terms of maternal age, maternal weight, and BMI. The mean CMT levels were 7.36 +/- 2.65% in the short-duration group and 10.65 +/- 5.07% in the long-lactation group. The mean energy contents were 3103.7 +/- 863.2 kJ/L in the short-duration group and 3683.2 +/- 1032.2 kJ/L in the long-duration group. The mean CMT levels and mean energy contents were correlated significantly with the duration of lactation (R2 = 0.22 and R2 = 0.23, respectively). In multivariate regression analysis, CMT levels (or energy contents) were not influenced by maternal age, diet, BMI, or number of daily feedings but remained significantly influenced by the duration of lactation. CONCLUSIONS Human milk expressed by mothers who have been lactating for >1 year has significantly increased fat and energy contents, compared with milk expressed by women who have been lactating for shorter periods. During prolonged lactation, the fat energy contribution of breast milk to the infant diet might be significant.