• Holder pasteurization (thermal processing) is the current processing standard for donor human milk (DHM). • Emerging alternatives include freeze-drying and subsequent low-dose gamma irradiation. • Volatile and protein profiles were analyzed to determine nutritional integrity after treatment. • Freeze-drying and low-dose gamma irradiation is a viable method to create an emergency reserve of safe donor human milk with long shelf life and thus security of supply during humanitarian crises. The availability of donor human milk (DHM) is currently limited by the volumes that can be thermally pasteurized and kept in long-term cold storage. This study assesses the application of freeze-drying followed by low-dose gamma irradiation of DHM for simplified, safe long-term storage. Solid-phase microextraction (SPME) GC–MS, SDS and native PAGE gel electrophoresis demonstrated that the overall changes in volatile and protein profiles in Holder pasteurized and freeze-dried DHM was negligible compared to the natural variations in DHM. Freeze-dried DHM samples (moisture < 2.2 %) processed with 2 kGy gamma irradiation did not show any significant lipid oxidation end-products and variation in protein profile. Therefore, freeze-drying followed by in-packaging gamma irradiation could be a safe method for pasteurization, convenient storage and delivery of DHM at ambient temperature. These methods may generate a means to create a reserve stock of DHM for emergencies and humanitarian aid. [ABSTRACT FROM AUTHOR]