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Fortifying human milk with protein and minerals appears to increase its susceptibility to bacterial growth, particularly under routine hospital nursery conditions. Bacterial counts, immune protein levels, and measurements of nutrient breakdown were analyzed in five fresh and 10 frozen fortified and unfortified human milk samples over a 72-hour storage period. Bacterial counts were higher in fortified milk compared to unfortified milk and increased over time. Similar measurements repeated on fortified milk under simulated nursery conditions revealed increased bacterial counts over a typical 4-hour use cycle., Objective. Data are scarce regarding the effects of nutrient fortification and storage on the immunoprotective properties of human milk. These effects are important considerations when feeding premature infants. We hypothesized that total bacterial colony counts (TBCC) and immunoglobulin A (IgA) concentration were not affected by the addition of fortifier even when tested under extreme storage conditions and that osmolality of fortified human milk does not increase with storage. Methods. Ten frozen and five fresh milk samples from mothers of premature infants were divided into fortified and unfortified milk, and stored for 72 hours at either refrigerator or room temperature. Aliquots were obtained at 0 to 72 hours for TBCC, osmolality, and total IgA, and analyzed by repeated measures analysis of variance (ANOVA). Results. [Log.sub.10] TBCC in milk stored at refrigerator temperature for 0, 24, 48, and 72 hours were significantly greater in fortified vs unfortified milk; both increased similarly with storage. Osmolality was greater in fortified than unfortified milk; both increased by approximately 4% with storage. IgA concentration was not affected by fortification or storage. To simulate the usual nursery use of fortified human milk, a separate evaluation was performed. Fortified milk was stored at refrigerator temperature for 20 hours, warmed in a 40 [degrees] C laboratory incubator for 20 minutes, and placed in a 34 [degrees] C infant incubator for 4 hours. Samples for TBCC were obtained at 0, 20, and 24 hours and analyzed by repeated measures ANOVA. [Log.sub.10] TBCC in fortified, refrigerated milk did not change over the 20-hour storage but increased during the simulated 4-hour usage. Conclusions. These findings may warrant consideration when using human milk in the neonatal nursery but support recommendations to use commercially fortified human milk within 24 hours. Pediatrics 1997;l00: 240-243; fortified human milk, immunoprotective properties, premature infants., ABBREVIATIONS. IgA, immunoglobulin A; TBCC, total bacterial colony counts; ANOVA, analysis of variance. Human milk is being used increasingly to feed the premature infant.[1] The high bioavailability of its nutrients [...]