Introduction Endurance training elicits tremendous adaptations of the mitochondrial energetic capacity. Yet, the effects of training or physical fitness on mitochondrial efficiency during exercise are still unclear. Accordingly, the purpose of the present study was to examine in vivo the differences in mitochondrial efficiency and ATP cost of contraction during exercise in two groups of adults differing in their aerobic capacity. Method We simultaneously assessed the ATP synthesis and O2 fluxes with 31P-magnetic resonance spectroscopy and pulmonary gas exchange measurements in seven endurance-trained (ET, V˙O2max: 67 ± 8 mL·min−1·kg−1) and seven recreationally active (RA, V˙O2max: 43 ± 7 mL·min−1·kg−1) subjects during 6 min of dynamic moderate-intensity knee extension. Results The ATP cost of dynamic contraction was not significantly different between ET and RA (P > 0.05). Similarly, end-exercise O2 consumption was not significantly different between groups (ET: 848 ± 155 mL·min−1 and RA: 760 ± 131 mL·min−1, P > 0.05). During the recovery period, the PCr offset time constant was significantly faster in ET compared with RA (ET: 32 ± 8 s and RA: 43 ± 10 s, P < 0.05), thus indicating an increased mitochondrial capacity for ATP synthesis in the quadriceps of ET. In contrast, the estimated mitochondrial efficiency during exercise was not significantly different (P/O, ET: 2.0 ± 1.0 and RA: 1.8 ± 0.4, P > 0.05). Consequently, the higher mitochondrial capacity for ATP synthesis in ET likely originated from an elevated mitochondrial volume density, mitochondria-specific respiratory capacity, and/or slower postexercise inactivation of oxidative phosphorylation by the parallel activation mechanism. Conclusion Together, these findings reveal that 1) mitochondrial and contractile efficiencies are unaltered by several years of endurance training in young adults, and 2) the training-induced improvement in mitochondrial energetic capacity appears to be independent from changes in mitochondrial coupling.