We investigated the effect of the composition of the storage container, Holder pasteurisation, and conditions during tube feeding on the concentration of selected vitamins in human milk. Though the fat soluble vitamins A, D, and E were not affected, the concentration of several water soluble vitamins decreased. The lower vitamin C concentration of milk stored in polypropylene containers compared with milk stored in glass containers (29%) was not significant. Holder pasteurisation significantly lowered the concentrations of vitamins C (36%), folacin (31%), and B6 (15%). Tube feeding significantly lowered the concentrations of vitamins C (44%) and B6 (19%), and exposure to phototherapy seemed to lower the vitamin C concentration (53%) further. Low birthweight infants have increased vitamin requirements. Vitamin losses in expressed human milk before or during feeding may increase the incidence of vitamin deficiencies in these babies.