There has been a notable increase in interest over recent years in the study of psychophysiological processes as they relate to the domains of sport and exercise. The development of psychophysiological techniques has been significant in the last 2 decades in the field. Their effective interconnection between the psychological and physiological dimensions makes them applicable across a variety of disciplines. Globally, psychophysiological measures are used to assess the degree of arousal or activation. Heart rate, skin conductance, pupillometry, and brain activity are all sensitive to a number of psychological constructs experienced by athletes, including workload, stress, anxiety, and fatigue. This chapter is concerned with techniques that facilitate the assessment of autonomic and somatic nervous system responses, as well as central nervous system responses that can be deployed during exercise and sport. These responses are based either on brain activity, as observed through mobile techniques such as scalp electroencephalography and functional near-infrared spectroscopy, or more generally on organic activity, as evidenced by changes in physiological responses such as electrodermal, pupillary, respiratory, muscular, or cardiac activity. Further investigation is still required to ascertain the impact of psychophysiological measures in real-world settings.