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Background and Importance Fortification of breast milk (BM) is routinely implemented to meet the nutritional requirements of preterm infants. Clinical guidelines recommend a safe osmolality range of 400–600 mOsm/kg for infant feeds. Exceeding this limit may increase the risk of gastrointestinal intolerance and necrotising enterocolitis in neonates.Aim and Objectives This study aimed to determine the osmolality of donated breast milk (DBM) from a tertiary hospital milk bank before and after fortification according to the manufacturer’s instructions, and to assess whether the resulting values exceeded the recommended safety range.Material and Methods DBM samples from three healthy donors were analysed in unfortified and fortified states. Fortification consisted of adding 1 g of fortifier per 25 mL of milk, as specified by the manufacturer. Osmolality was measured in duplicate at 0, 4, 8, 12, 16, 20 and 24 hours after preparation using a freezing-point osmometer. Samples were stored under refrigeration throughout the study period. Data were obtained from the milk bank traceability system and analysed with statistical software.The study was conducted in collaboration with the Pharmacy, Neonatology, and Clinical Analysis Departments of the hospital.Results A total of six samples (84 measurements) were analysed.The mean osmolality of unfortified DBM at t = 0 h was 299.3 ± 4.2 mOsm/kg, increasing slightly to 301.0 ± 3.5 mOsm/kg after 24 h.Fortified DBM showed higher values: 385.7 ± 7.8 mOsm/kg at t = 0 h, rising to 417.0 ± 8.2 mOsm/kg after 24 h.Osmolality increased significantly after fortification (p < 0.001). During storage, unfortified DBM remained stable (p = 0.59), whereas fortified samples showed a significant increase (p = 0.009).Conclusion and Relevance Fortifier addition significantly increased human milk osmolality, although all values remained within the recommended safety range. Storage for up to 24 hours led to a further osmolality increase only in fortified samples. Mean post-fortification osmolality was slightly higher than that reported by the manufacturer. Larger studies are warranted to confirm these findings and to further investigate the effect of storage time on osmolality.Conflict of Interest No conflict of interest