Exercise causes a dramatic increase in energy requirements because of the metabolic needs of working muscles. Exercise-dependent factors regulate fuel use. Absolute exercise intensity determines the exercise-induced increase in energy demands, whereas exercise intensity relative to an individual's maximal aerobic capacity (VO2max) determines the proportional contribution of different fuel sources (i.e. plasma glucose, plasma fatty acids, muscle glycogen and intramuscular triacylglycerols). Endurance training increases aerobic capacity in muscle and the oxidation of fat during exercise. In addition, exercise-independent factors, such as diet composition, sex, age, and body composition also influence substrate use during exercise. The present review discusses the regulation of substrate use during exercise in human subjects, with a focus on the role of exercise-independent factors.