This study was aimed at evaluating the effect of prolonged refrigeration of expressed human milk on its protein and microbial profile. Human milk from mothers of pre-term newborns was collected, pooled and stored in the Neonatal Intensive Care Unit refrigerator. Pooled milk was aliquoted and analysed within 96 h. The milk samples were analysed for pH, total protein content, protein profile, secretory immunoglobulin A (sIgA), available lysine and lactose contents, and bacterial profile (total aerobic bacterial count, Enterobacteriaceae, coagulase-positive Staphylococci, lactic acid bacteria). The refrigeration process did not affect the abundance of single proteins, nor the available lysine and sIgA contents. Lactose and pH showed a significant, although limited, decrease. The main microbial communities of human milk were preserved during refrigerated storage. Important markers of milk nutritional quality and bioactive proteins, including the antimicrobial components lysozyme and lactoferrin, as well as immunological components, were preserved. (C) 2013 Elsevier Ltd. All rights reserved.