Background: It is well known that breast-feeding protects the newborn from infectious diseases. This is especially important for very low birth weight preterm infants, whose immune systems are immature. In this study we investigated how a milk fortifier and replacement formula affected lymphocyte subsets in preterm infants. Method: The study assessed the effects of different types of feeding (human milk, n = 14; fortified human milk, n = 16; formula, n = 14) on lymphocyte subsets in 44 very low birth weight preterm infants. For each baby, two consecutive blood samples were collected 7–10 days apart during the full enteral feeding period. For each sample, the percentages of CD3+ (pan-T), CD19+ (B-cell), CD4+ (T-helper), CD8+ (T-suppressor), and CD3–CD16/56+ (natural killer cell) lymphocytes were measured in a flow cytometer, and the absolute count for each subset was calculated based on the total lymphocyte count. Within each feeding group, the absolute numbers of each lymphocyte subset in the two consecutive samples were compared. Also, the mean absolute counts for each cell type were compared among the 3 groups for the first set of blood samples, and the same comparisons were made for the second set. Results: The mean number of CD3–CD16/56+ cells in the formula-fed infants was significantly lower than the corresponding means in the groups fed human milk alone and fortified human milk (p = 0.037). Conclusion: The findings suggest that babies fed formula have different lymphocyte subset compositions than those fed breast milk or fortified breast milk.