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Annual killifish are among the most remarkable extremophile species with the shortest vertebrate life span. Few studies have reported on the oxidative balance throughout their life cycle and its association to the natural aging process of these neotropical animals in a natural environment. We standardized and analyzed physiological markers related to the redox balance of the annual killifish (Cynopoecilus fulgens) throughout the post-embryonic life cycle (enzyme activity of Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione S-transferase, as well as the determination of the levels of Lipoperoxidation, Carbonylated Proteins, and Total Proteins). We tested the influence of environmental variables on these biomarkers. Individuals were collected, including juveniles, adults, and seniles, in three sampling units around the Parque Nacional da Lagoa do Peixe, located in the Coastal Plain of Rio Grande do Sul. We observed that males and females used different physiological strategies of their redox balance during their life cycle, and their oxidative balance was influenced by their reproductive period and environmental variables (water temperature, abundance of predators, abundance of another sympatric annual killifish species, and abundance of C. fulgens). The population of each temporary pond presented different physiological responses to the adaptation of their life cycle, and there was an influence of environmental component as a modulator of this cycle. Our study offers reference values that will be useful for comparison in future research with short-lived organisms.