Abstract Objective To identify changes in macronutrient content of very preterm human milk associated with perinatal factors. Study design Milk macronutrients were measured on weeks 1, 2, 4 and 8 with mid-infrared transmission spectrometers. Result We assessed 625 samples (from 117 mothers and 130 very preterm infants). Average concentrations were: protein 1.3 ± 0.3 g/dl, carbohydrates 7.3 ± 0.6 g/dl, fat 3.7 ± 1.0 g/dl and energy 296.0 ± 41.0 kJ/dl (70.7 kcal/dl). Gestational age negatively correlated with protein (rho: −0.307, p < 0.001) and energy (r: −0.193, p = 0.003). Advanced maternal age, gestational age and intrauterine growth restriction were independently associated with milk protein content over the first 4 weeks (adjusted R²: 0.113, p = 0.002). Conclusion These findings may help neonatologists identify patients fed Mother´s Own Milk who are at increased risk of poor postnatal growth.