In healthy individuals maximal oxygen uptake is limited by cardiac output when exercising with large musclemass, e.g. 2-legged cycling (BIKE), whereas during one-leg knee extension (KE) exercise there is a closermatch between the amounts of O2 delivered to O2 demand. However hyperoxia has been shown to increaseO2 uptake in both work modalities. This study examined the extent to which hyperoxia affects leg oxygentransport, muscle diffusion capacity, mitochondrial oxygen affinity (p50mito) in vivo and ex vivo, and muscleO2 uptake during exercise engaging a large and small muscle mass in well-trained individuals. In this studywe show that light hyperoxia increases O2 uptake at peak incremental exercise (~10%, p< 0.05) in bothBIKE and KE due to an increased O2 delivery (~ 10%, p< 0.05), which in turn is mainly caused by anincreased arterial O2 content (~5%, p< 0.05), and a non-significant increased leg blood flow (~ 5%). Hencehyperoxia decreases mitochondrial O2 affinity as indicated by the higher calculated in vivo mitochondrialp50. Inspired O2 enhanced maximal work rate in BIKE (~6.5%) and in KE (~4.5%).Our data show that in vivo mitop50 is lower than the ex vivo measure in BIKE, whereas in vivo mitop50 issimilar to ex vivo measure in KE. These indicates that mitochondrial OXPHOS capacity was in excess inBIKE, whereas in KE was almost fully utilized in vivo. This study indicated that both during whole body peakexercise as well as during exercise with smaller muscle mass, V̇O2 may not limit O2 diffusion capacity.