Summary:Protein (P), fat (F), and carbohydrate (C) concentration in expressed human bank milk was determined by infrared analysis of 2,554 samples from 224 mothers. The mean contents of P, F, C, and energy (E, calculated from P, F, and C) were 9.0 g/L, 39.0 g/L, 71.9 g/L, and 696 kcal/L, respectively. There was a large variation in the concentration of energy‐yielding macronutrients. The contents of P. F, C, and E in the samples with the highest values (97.5 percentile) were 2.3‐, 4.8‐, 1.2‐, and 2.3‐fold, respectively, above the contents in the samples with the lowest values (2.5 percentile). The P content decreased exponentially during the 1st 8 months, followed by an increase during the following months. The F content decreased during the 1st 4 months, followed by an almost linear increase. The possible influence of different maternal characteristics on the macronutrient content of the milk was examined. The main results were as follows: the P and F contents increased slightly with increasing body mass index of the mother, the P content decreased with increasing amounts of milk delivered to the milk bank, and the F content was higher in mothers delivering large amounts of milk. By selecting incoming milk with a high P content, we have developed a “high‐protein” milk with a P content of about 12 g/L (true protein) and an E content of about 725 kcal/L. Thus, by continuous monitoring of macronutrient content in human bank milk it is possible to develop a “high‐protein” milk with sufficient P and E content to cover the needs of preterm infants with very low birth weights (< 1,500 g).