Introduction: The macronutrient concentrations of human milk could be influenced by the various processes used in human milk bank. Aims: To determine the effect of various process (Holder pasteurization, freezing and thawing and feeding method) on the macronutrient concentration of human milk. Methods: The samples of donated fresh human milk were studied before and after each process (Holder pasteurization, freezing and thawing and feeding method) until their delivery to newborn infants. Fifty-seven raw human milk samples were analyzed in the first step (pasteurization) and 228 in the offer step. Repeated measurements of protein, fat and lactose amounts were made in samples of human milk using an Infrared analyzer. The influence of repeated processes on the mean concentration of macronutrients in donor human milk was analyzed by repeated measurements ANOVA, using R statistical package. Results: The most variable macronutrient concentration in the analyzed samples was fat (reduction of 59%). There was a significant reduction of fat and protein mean concentrations following pasteurization (5.5 and 3.9%, respectively). The speed at which the milk was thawed didn't cause a significant variation in the macronutrients concentrations. However, the continuous infusion delivery significantly reduced the fat concentration. When the influence of repeated processes was analyzed, the fat and protein concentrations varied significantly (reduction of 56.6% and 10.1% respectively) (Pb0.05). Lactose didn't suffer significant reductions in all steps. Conclusion: The repeated processes that donor human milk is submitted before delivery to newborn infants cause a reduction in the fat and protein concentration. The magnitude of this decrease is higher on the fat concentration and it needs to be considered when this processed milk is used to feed preterm infants.