The measurement of substrate oxidation in living individuals can facilitate metabolic investigations. Indirect calorimetry and tracer techniques allow such measurements. Indirect calorimetry provides simultaneous calculation of the rate of oxidation of the three major macronutrients (carbohydrates, fat and protein) from respiratory gas exchanges and urinary nitrogen excretion. Such estimates represent net substrate oxidation rates. Thus, carbohydrate oxidation represents the oxidation of either endogenous glycogen or exogenously administered carbohydrate. It also includes de novo lipogenesis (with simultaneous oxidation of lipids in amounts equivalent to their synthesis), but does not include oxidation of glucose formed from gluconeogenesis from amino acids or glycerol. The accuracy of these calculations depends on the adequacy of the stoichiometry used for oxidation of substrates, which has to be varied when special forms of substrate are used. Tracer techniques consist in administration in tracer amounts of a selected nutrient labelled with 14C or 13C and monitoring of the specific activity/isotopic enrichment of the substrate in plasma and of the pulmonary elimination of labelled CO2. Such techniques allow assessment of the actual rate of oxidation of one substrate at a time. However, there are major pitfalls relating to the recovery of labelled CO2 in breath during oxidation of the substrate as well as during non-steady state conditions.