To determine the effects of age and sex on in vivo mitochondrial function of distinct locomotory muscles, the tibialis anterior (TA) and medial gastrocnemius (MG), of young (Y; 24±3 years) and older (O; 69±4) men (M) and women (W) of similar overall physical activity (PA) was compared. In vivo mitochondrial function was measured using phosphorus magnetic resonance spectroscopy, and PA and physical function were measured in all subjects. Overall PA was similar among the groups, although O (n=17) had fewer daily minutes of moderate-to-vigorous PA (p=0.001), and slowed physical function (p<0.05 for all variables), compared with Y (n=17). In TA, oxidative capacity (V max ; mM s −1 ) was higher in O than Y (p<0.001; Y= 0.90±0.12; O=1.12±0.18). There was no effect of age in MG (p =0.5; Y= 0.91 ± 0.17; O = 0.96 ± 0.24), but women had higher oxidative capacity than men (p= 0.007; M=0.84±0.18; W=1.03±0.18). In vivo mitochondrial function was preserved in healthy O men and women, despite lower intensity PA and physical function in this group. The extent to which compensatory changes in gait may be responsible for this preservation warrants further investigation. Furthermore, women had higher oxidative capacity in the MG, but not the TA.