Abstract. Objective: To compare the effects of four thawing methods on the fat concentration, free fatty acid concentration, total antioxidant capacity, and epidermal growth factor concentration of frozen human milk. Study design: Milk samples were obtained from 16 lactating mothers who delivered prematurely. Milk samples were divided into aliquots and frozen at −20◦C. Aliquots were thawed at 4◦C (refrigerator), 20◦C (room temperature), 50◦C (hot water) or microwaved. After thawing, the aliquots were analyzed for fat, free fatty acid concentration, total antioxidant capacity, and epidermal growth factor concentration. Results: The mean fat concentration (±SD) was 6.0 ± 1.8 (4◦C), 5.9 ± 2.3 (20◦C), 4.9 ± 1.8 (50◦C), and 4.1 ± 1.8 (microwave), P < 0.001. The free fatty acid concentration was 4.0 ± 1.1 (4◦C), 3.6 ± 1.4 (20◦C), 2.9 ± 1.5 (50◦C), and 2.4 ± 1.4 nmol/l (microwave), P < 0.001. The total antioxidant capacity was 8.8 ± 1.2 (4◦C), 8.2 ± 1.2 (20◦C), 7.9 ± 1.5 (50◦C), and 7.1 ± 1.1 nmol/l (microwave), P < 0.001. No statistically signiﬁcant change in epidermal growth factor concentration was noted. Conclusions: Thawing at 4◦C and 20◦C had the least effects with microwaving having the most effects on human milk’s fat concentration, free fatty acid concentration, and total antioxidant capacity. Epidermal growth factor concentration was not affected by the thawing methods.