Background: Microwave heating can be effective in preventing cytomegalovirus infection transmitted via human milk. Temperature changes during microwaving using different containers, in different areas inside a container, or using milk from different mothers are not well studied. Research Aim: To determine temperature changes of human milk during microwaving using different containers, in different container areas, using different human milk, and in a 30-ml soft polypropylene bag (sachet) immersed in water. Methods: In this experimental in vitro study, human milk (100 ml) was poured into six different bottles. The temperature was monitored simultaneously at each bottle’s bottom and surface (microwaving at 600 W) and at nine places inside the container (microwaving at 500 W). Human milk (20 ml) from six participants was inserted into a sachet, then immersed in 80 ml of human milk or water in a bottle, and the temperatures inside and outside the sachet during microwaving (at 500 W) were monitored. Results: The temperature changes at the surface were significantly larger than those at the bottom. Temperatures at the bottoms of different bottles, of human milk from different participants, or inside and outside the sachet, did not differ significantly. No temperature outliers inside the bottle were observed. Conclusion: Microwaving at 500 W and 600 W for 60 s was not significantly different in temperature changes among different areas inside bottles or among milk from different participants. A small volume of human milk (up to 100 mL) can be heated using a sachet.