Introduction: Exclusive breastfeeding, especially in the first six months, is essential for infants as it provides nutrition and protection against various diseases. Colostrum, which is found in the first breast milk produced, contains various protective factors, such as lactoferrin and lysozyme. Human milk can be stored at room temperature, refrigerated, or kept frozen. Several factors affect the stability of the bioactive content in human milk, such as temperature and storage time. The aim of this study was to measure the stability of lactoferrin and lysozyme levels in human milk during the first six hours (h) at different temperatures and compare it with that of frozen human milk. Methods: Human milk samples were obtained from 11 breastfeeding mothers using certain criteria. The human milk was stored at room temperature and 4°C for 1, 3, and 6 h and classified as never frozen, while frozen human milk was stored at -20°C for 1, 3, and 6 days. Measurement of the lactoferrin and lysozyme levels was performed using enzyme-linked immunosorbent assay. Results: The results showed that storage at room temperature significantly reduced lactoferrin and lysozyme levels. Lactoferrin levels in frozen human milk did not significantly decrease during the first six days. Meanwhile, the lysozyme levels in frozen human milk decreased significantly. Conclusion: The levels of lactoferrin and lysozyme in frozen human milk stored for the first six days were more stable than those stored at room temperature and 4°C in the first 6 h.