Sedentary behaviours—too much sitting as distinct from too little exercise—are emerging as a ubiquitous, modern-day health hazard. Epidemiological evidence is accumulating that indicates greater time spent in sedentary behaviour is associated with increased cardiometabolic risk, even when controlling for the influence of leisure time moderate-to-vigorous physical activity. Based on these observations and preliminary experimental work, it has been proposed that sedentary behaviour influences health risk in part through some distinct mechanisms that act independently of lack of physical activity. However, the observational evidence is well ahead of evidence on physiological responses and potential biological mechanisms that may underlie the observed associations. Here, we summarize and discuss experimental evidence to date on the physiological effects of sedentary behaviours (prolonged sitting), including potential countermeasures aiming to address too much sitting as a health risk. We also highlight future research that is needed to further ascertain the impact of sedentary behaviour on altering physiology.