Astronauts undertaking long-duration space missions may be vulnerable to unique stressors that can impact human aging. Nevertheless, few studies have examined the relationship of mission duration with DNA-methylation-based biomarkers of aging in astronauts. Using data from the six participants of the Mars-500 mission, a high-fidelity 520-day ground simulation experiment, we tested relationships of mission duration with five longitudinally measured blood DNA-methylation-based metrics: DNAmGrimAge, DNAmPhenoAge, DNA-methylation-based estimator of telomere length (DNAmTL), mitotic divisions (epigenetic mitotic clock [epiTOC2]), and pace of aging (PoA). We provide evidence that, relative to baseline, mission duration was associated with significant decreases in epigenetic aging. However, only decreases in DNAmPhenoAge remained significant 7 days post-mission. We also observed significant changes in estimated proportions of plasmablasts, CD4T, CD8 naive, and natural killer (NK) cells. Only decreases in NK cells remained significant post-mission. If confirmed more broadly, these findings contribute insights to improve the understanding of the biological aging implications for individuals experiencing long-duration space travel.