Freezing at -80 °C significantly decreases the energy content of HM, both from fat and carbohydrates. Since quantitatively the decrease in macronutrients was much higher than that published for HM storage at -20 °C, our results do not support freezing HM at -80 °C as the gold standard for long-term storage. We suggest that caloric intake calculations in preterm infants cannot be established based upon fresh HM data.

Circadian variations in fat and energy concentrations of HM are consistent over the first 7 weeks of lactation. There are no consistent circadian variations in HM protein and carbohydrates. Over a given day, there are little variations in protein and carbohydrates content, but fat concentrations are more variable, and evening values are less well predicted by morning sample analysis than values for protein or carbohydrates.

Manually expressed human milk had higher fat content than milk expressed by electric pump. We speculate that this difference is due to the presence of hindmilk in the manually expressed milk because the technique of massaging the breast during manual expression is more likely than the pump to eject hindmilk, which has been shown to have higher fat content than foremilk.