Background Human milk is the optimal source of nutrition for preterm infants. However, breast milk alone is often not sufficient to satisfy the high nutritional needs for growth and development in preterm infants. Fortified human breast milk is the best way to meet the nutritional needs of preterm infants. Human breast milk is fortified according to the estimated nutrient content of mature breast milk; however, because the content of breast milk is highly variable, the macronutrient support may be more or less than needed. The goal of this study was to analyze the macronutrient content of preterm human milk during the first 6 weeks of lactation. Methods The study included 32 mothers of preterm infants with a gestational age of ≤32 weeks. Breast milk was collected in 24-h cycles and analyzed daily using mid-infrared (MIR) spectroscopy. We measured protein, fat and lactose concentrations in the breast milk, and the energy content was calculated. Results The protein content was high during the first weeks of lactation, but decreased as lactation progressed. The fat, energy and lactose contents of the breast milk were low during the first 2 weeks of lactation, increased as lactation progressed and remained constant thereafter. In women with high body mass index (BMI), higher protein levels were found in transitional milk. In women who had high income level, higher fat and energy levels were found in transitional milk. Conclusion Our findings indicate that the macronutrient content of preterm breast milk changes throughout the course of lactation, with BMI and income level. Knowledge of the macronutrient composition of breast milk is necessary to ensure that preterm infants receive the appropriate types and quantities of nutrients to promote optimal growth, and to ensure that breast milk is fortified according to individual needs. Our findings may be useful for the provision of optimal nutritional support for preterm infants.