Abstract Aging reduces the functional competence of T cells. T cell metabolism regulates their function, with mitochondrial defects in mice resulting in aged phenotypes, including accelerated senescence. Physical activity (PA) maintains T cell function in older adults, although the mechanisms underlying this effect are poorly understood. This study examined the effects of aging on the metabolic profile of T cell subsets and investigated whether PA could improve metabolic function in T cells from older donors. We recruited nine young adults (23 ± 3y) and 19 healthy older adults who had high PA (HPA, N=9, 75.5 ± 4.7y) or low PA levels (LPA, N=10, 76.4 ± 2.1y), based on their moderate-to-vigorous PA scores. We investigated the metabolic profiles of CD4+ and CD8+ T cells at rest and post-activation (PMA and ionomycin), via SCENITH flow cytometry. Compared to young adults, older adults had higher mitochondrial dependence (MD) in unstimulated CD4+ and CD8+ naive, effector memory (EM) and central memory (CM); and higher protein synthesis in CD4+ EM, CD4+ naïve, CD8+ EM, suggesting higher energetic demand in T cells with aging. Upon activation there was a lower reduction in MD of CD4+ EMRA and CD8+ EMRA; and a greater increase in IL-6 and TNFα expression in CD8+ cells of older than young adults, indicative of impaired metabolic flexibility with aging. PA effects were more prominent in unstimulated CD8+ cells, where HPA had lower glucose dependence (GD) for overall CD8+, CD8+EM and a trend to higher MD in CD8+ CM than LPA. Upon activation, HPA had a lower increase in CD4+ TNFα expression and trended to have a higher reduction in MD of overall CD4+ and a higher reduction in GD of CD4+ EMRA, than LPA. This suggests a lower metabolic demand in CD4+ T cells of HPA. We concluded that PA could modify T cell metabolic profile at rest, and following activation, in older adults, which may explain the better T cell function in physically active older individuals.