One of many problems to be faced when assessing in vivo human muscle mitochondria respiration by phosphorus magnetic resonance spectroscopy (31P-MRS) is the definition of the correct reference population and the values of reference range. To take into account most factors that influence muscle activity as age, sex, physical activity; nutritional state etc., an exceedingly high number of different reference groups are needed. To overcome this problem we developed specific tests to assess separately in vivo the activity and the functionality of muscle mitochondria by 31P-MRS in clinical settings. By activity we refer to muscle whole metabolic activity, i.e. the total oxidative capacity of muscle mitochondria which is influenced by many factors (age, sex, physical activity, nutritional state etc.). By functionality we refer to the qualitative aspects of mitochondrial respiration which depends on the integrity of mitochondrial multienzyme systems and on substrate availability. Our tests have been experienced on some 1200 patients and are currently used to detect deficits of mitochondrial respiration and ion transport in patients with suspected primary or secondary muscle mitochondrial malfunctioning.