Stoichiometric indirect calorimetry is the most commonly used method for assessing the oxidation of fatty acids (FAO) and carbohydrates (CHO). It relies on gaseous exchange measurements of oxygen uptake (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) for accurate estimations of FAO and CHO. During exercise protein utilization contributes minimally compared with FAO and CHO contribution. Hence, common mathematical estimations are based on FAO and CHO relative contribution at various exercise intensity domains. The utilization of FAO predominates at low exercise intensities, and its relative contribution gradually decreases as exercise intensity increase in favour of CHO contribution. Higher exercise intensities beyond RER ≥1 reflect an excess non-oxidative CO2 from bicarbonate buffering, which further elevates VCO2 and caused an overestimated CHO and underestimated FAO. Therefore, detecting meaningful effects on FAO is often measured below the severe exercise intensity domain and corresponds to exercise intensities below approximately 85% of maximal VO2 (VO2 max). The most common diagnostic indices derived from FAO, CHO and corresponding power output include: 1) maximal fat oxidation (MFO), thought to correspond to approximately 30-75% of VO2 max, and is defined as the power output or exercise intensity at which FAO becomes maximal. 2) the crossover point (COP), defined as the power output at which energy expenditure at which energy derived from CHO predominates over that from FAO. Prolonged single intensity, testing protocols have long been shown to provide a valid estimate of FAO and CHO because they allow a steady state attainment for the gaseous exchange attainment. However, they
require several laboratory visits, and so they can be less practical compared with the incremental exercise protocols commonly being used. However, it is important that incremental protocols consider the selection of an appropriate initial workload, stage increment, and stage duration.