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To examine the integrity (pH, bacterial counts, host defense factors, nutrient contents, and osmolality) of freshly expressed and previously refrigerated human milk subjected to long-term freezer storage.Mothers donated 100 mL of freshly expressed milk. Samples were divided into baseline, storage at −20°C (fresh frozen) for 1, 3, 6, and 9 months, and prior storage at +4°C for 72 hours (refrigerated frozen) before storage at −20°C for 1 to 9 months. Samples were analyzed for pH, total bacterial colony count, gram-positive and gram-negative colony counts, and concentrations of total protein, fat, nonesterified fatty acids, lactoferrin, secretory IgA, and osmolality.Milk pH, total bacterial colony count, and Gram-positive colony counts decreased significantly with freezer storage (P < .001); bacterial counts decreased most rapidly in the refrigerated frozen group. The gram-negative colony count decreased significantly over time (P < .001). Nonesterified fatty acid concentrations increased significantly with time in storage (P < .001). Freezing for up to 9 months did not affect total protein, fat, lactoferrin, secretory IgA, or osmolality in either group.Freezer storage of human milk for 9 months at −20°C is associated with decreasing pH and bacterial counts, but preservation of key macronutrients and immunoactive components, with or without prior refrigeration for 72 hours. These data support current guidelines for freezer storage of human milk for up to 9 months for both freshly expressed and refrigerated milk.