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We aim to assess the nutritional composition of shelf-stable (SS) human milk and compare the nutritional profile to Holder pasteurized (HP) and raw human milk from the same pool.Milk samples from 60 mothers were pooled. From this pool, 36 samples were taken; 12 samples were kept raw, 12 samples were HP, and 12 samples were retort processed to create an SS product. Samples were analyzed for percent fat, percent solids, total protein, lactose, amino acids, and thiamine.Percent fat, percent solids, and lactose were similar between raw, HP, and SS samples. Total protein was statistically increased in SS samples when compared to raw (P = 0.005) and HP (P < 0.001) samples, but protein differences were not clinically relevant (raw = 15.1 mg/mL, HP = 14.8 mg/mL, and SS = 15.8 mg/mL). Lysine was the only amino acid impacted by processing, and its destruction increased as heat increased (raw = 0.85 mg/100 mL, HP = 0.77 mg/100 mL, SS = 0.68 mg/100 mL). Total thiamine was significantly decreased in SS samples (0.14 mg/L; P < 0.01) when compared with raw samples (0.24 mg/L) and HP samples (0.26 mg/L).Macronutrient content is relatively unaffected by processing; Holder pasteurization and retort processing maintain similar fat, lactose, and total protein levels. Lysine and thiamine were significantly decreased by retort processing, but not by Holder pasteurization. Thiamine losses are clinically significant, and fortification may be necessary if SS donor milk is a long-term feeding choice.