Chronic exercise improves endurance and skeletal muscle oxidative capacity. Despite the potential importance of reactive oxygen species (ROS) generated during exercise as regulators of these adaptations, the effect of repeated bouts of aerobic exercise on ROS generation by skeletal muscles during contractions has not been examined. Our aim was to establish the impact of repeated treadmill running exercise on muscle ROS generation and activation of redox-sensitive transcription factors. Following 8 weeks of treadmill running, mice displayed an improvement in running speed that was associated with an enhanced ability of gastrocnemius (GTN) muscles to maintain force during a protocol of isometric contractions. In contrast to GTN muscles of cage-sedentary (Sed) mice, muscles from exercised (Exer) mice did not release superoxide or nitric oxide during the isometric contractions. For male mice, basal levels of nuclear factor κB (NFκB) and activator protein-1 (AP-1) DNA binding were increased by treadmill running, and the contraction-induced activation of NFκB and AP-1 observed in muscles of Sed mice was absent in Exer muscles. Also in contrast to Sed muscles, Exer muscles displayed no reductions in glutathione or protein thiol levels in response to contraction. Our observations of decreases for Exer compared with Sed muscles in contraction-induced (i) ROS generation, (ii) activation of redox-sensitive signalling pathways, and (iii) ROS stress suggest that exercise conditioning enhances the ability of skeletal muscle to readily and rapidly detoxify ROS and/or reduces ROS generation, providing protection from ROS-induced damage and reducing signals that might act to mediate further unnecessary adaptations.