During exercise, muscle ATP demand increases with intensity, and at the highest power output, ATP consumption may increase more than 100-fold above the resting level. The rate of mitochondrial ATP production during exercise depends on the availability of O2, carbon substrates, reducing equivalents, ADP, Pi, free creatine, and Ca2+. It may also be modulated by acidosis, nitric oxide and reactive oxygen and nitrogen species (RONS). During fatiguing and repeated sprint exercise, RONS production may cause oxidative stress and damage to cellular structures and may reduce mitochondrial efficiency. Human studies indicate that the relatively low mitochondrial respiratory rates observed during sprint exercise are not due to lack of O2, or insufficient provision of Ca2+, reduced equivalents or carbon substrates, being a suboptimal stimulation by ADP the most plausible explanation. Recent in vitro studies with isolated skeletal muscle mitochondria, studied in conditions mimicking different exercise intensities, indicate that ROS production during aerobic exercise amounts to 1-2 orders of magnitude lower than previously thought. In this review, we will focus on the mechanisms regulating mitochondrial respiration, particularly during high-intensity exercise. We will analyze the factors that limit mitochondrial respiration and those that determine mitochondrial efficiency during exercise. Lastly, the differences in mitochondrial respiration between men and women will be addressed.