Objective: This study aimed to evaluate the effect of milk expression method (manual expression versus electric pump) on the composition of breastmilk. Study Design: Data on 21 mothers of 21 newborns 48-72 hours postdelivery were collected and analyzed. The women were randomly assigned to express breastmilk manually followed by pump, or in reverse order. The fat, carbohydrate, and protein contents of the milk samples were analyzed using a human milk analyzer (Miris AB, Uppsala, Sweden). Results: The fat and energy contents of milk obtained through manual expression were higher than those obtained by pump (p=0.024 and p=0.04, respectively, by the Wilcoxon signed rank test). There were no significant differences in protein or carbohydrate content of milk obtained by either method of expression. The difference in fat content between milk obtained by the two methods was not correlated with mother's age, delivery method, gestational age at delivery, parity, or the interval between delivery and the time the sampled milk was obtained. Conclusions: Manually expressed human milk had higher fat content than milk expressed by electric pump. We speculate that this difference is due to the presence of hindmilk in the manually expressed milk because the technique of massaging the breast during manual expression is more likely than the pump to eject hindmilk, which has been shown to have higher fat content than foremilk.