As a result of concerns over the transmission of infectious diseases by donor milk, as well as the possible loss of nutritional value of donor milk through exposure to a variety of environmental conditions, the practice in the United States has been to discard unpasteurized donor milk that has thawed or sat for several hours at room temperature or in the refrigerator rather than (re)freezing it. We undertook an experiment to measure the effects of ambient temperature conditions and refreezing on the bioburden and nutritional content of human milk. We conclude that unpasteurized human milk is robust and can be used after storage under certain conditions.