In the last 30 years, the role of reactive oxygen species (ROS) in exercise physiology has received considerable attention. Acute physical exertion has been shown to induce an augmented generation of ROS in skeletal muscle via different mechanisms. There is evidence that ROS formation in response to vigorous physical exertion can result in oxidative stress. More recent research has revealed the important role of ROS as signaling molecules. ROS modulate contractile function in unfatigued and fatigued skeletal muscle. Furthermore, involvement of ROS in the modulation of gene expression via redox-sensitive transcription pathways represents an important regulatory mechanism, which has been suggested to be involved in the process of training adaptation. In this context, the adaptation of endogenous antioxidant systems in response to regular training reflects a potential mechanism responsible for augmented tolerance of skeletal muscle to exercise-induced stress. The present review outlines current knowledge and more recent findings in this area by focussing on major sources of ROS production, oxidative stress, tissue damage, contractile force, and redox-regulated gene expression in exercising skeletal muscle.