Background: Human milk is an essential source of nutrition for an infant’s health. When breastfeeding working mothers or students, for example, are unable to breastfeed, storing their milk is recommended. Therefore, it is crucial to know the storage conditions to ensure their antioxidant capacity and avoid oxidative damage. Research Aim: To evaluate the stability of the antioxidant and pro-oxidant profiles and the amount of total protein in fresh human milk after different storage times (1, 2, 7, 14, and 21 days) and temperatures (4 ºC and -20 ºC). Methods: This was a prospective, longitudinal, and observational study with milk samples grouped according to age for comparisons, which included 20 lactating women. The antioxidant activity was evaluated using the colorimetric methods of free radical scavenging 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid and the decrease of ferric ion. Oxidative stress was determined by the lipid peroxidation product formation through malondialdehyde concentration, and the total protein content was assessed by the Bradford method. Results: The antioxidant profile of human milk was maintained with minimal losses until the 14th day when stored at 4 ºC and -20 ºC. The evolution of malondialdehyde concentration over storage revealed significant changes only 21 days after human milk storage at 4 ºC. There was no change in the value of total protein content. Conclusions: To sum up, there is no difference in the storage of human milk at a temperature of 4 °C or -20 °C over 14 days. Therefore, the lactating woman may choose the most convenient way of storage.