High-temperature short-time (HTST, 72°C, 15 s) pasteurization can be successfully used for human milk (HM) preservation. The purpose of this study was to compare the effects of different HTST heating methods: microwave (MHTST) and standard convective (CHTST) on macronutrients, fatty acids (FAs), lactoferrin (LF), vitamin C, lysozyme (LZ), α-amylase (α-A) activity, lipid oxidation and antioxidant properties of HM. Regardless of the heating methods, the macronutrients and FA content, LZ activity and antioxidant properties of HM proved to be insensitive to HTST treatment, while the concentration of vitamin C decreased by 42.6% and 50.2%, and the activity of α-A by 6.0 and 7.2% when using MHTST and CHTST, respectively. LF content changed significantly only when CHTST was used (42.3% reduction). There were no negative effects of microwave heating under controlled conditions on the HM components. Satisfactory results regarding the impact of MHTST on HM composition and potential economic benefits suggest that this technology can be applied in HM banks to ensure adequate safety and quality.