Abstract. OBJECTIVE: To use real-time human milk macronutrient analysis to calculate final composition following fortification. STUDY DESIGN: Preterm HM (PHM) and pooled donor human milks (DHM) were analyzed by mid-infrared spectroscopy for protein, fat and lactose. Energy content was calculated from macronutrient results. Three lactation stages were constructed. DHM was compared to PHM. Four milk sample profiles were selected to demonstrate individualized fortification results. RESULTS: Lactose was similar in PHM and DHM. Protein in PHM showed the expected decline as lactation progressed. DHM protein was significantly lower vs. PHM. Fat was highly variable and lowest in DHM. Using standard fortification protocols, not all fortified milks met targets for protein and energy. Individualized fortification resulted in milks closer to target recommendations. CONCLUSIONS: Real-time analysis of HM provides assessment of the macronutrient content of the milk and can guide fortification. Individualized protocols, based on actual milk macronutrient profiles, may need to be considered to avoid unexpected nutrient content.