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Plasma free fatty acids (FFA) are a major contributor to whole body fat oxidation during exercise. However, the extent to which manipulating plasma FFA concentrations will influence whole body peak fat oxidation rate (PFO) during exercise remains elusive. In this study we aimed to increase plasma FFA concentrations through a combination of fasting and repeated exercise bouts. We hypothesized that an increase in plasma FFA concentration would increase PFO in a dose-dependent manner. Ten healthy young (31 ± 6 yr) (mean ± SD) well-trained (maximal oxygen uptake 65.9 ± 6.1 ml·min −1 ·kg −1 ) men performed four graded exercise tests (GXTs) on 1 day. The GXTs were interspersed by 4 h of bed rest. This was conducted either in a fasted state or with the consumption of a standardized carbohydrate-rich meal 3.5 h before each GXT. Fasting and previous GXTs resulted in a gradual increase in PFO from 0.63 ± 0.18 g/min after an overnight fast (10 h) to 0.93 ± 0.17 g/min after ∼22 h of fasting and three previous GXTs. This increase in PFO coincided with an increase in plasma FFA concentrations ( r 2  = 0.73, P &lt; 0.0001). Ingestion of a carbohydrate-rich meal 3.5 h before each GXT resulted in unaltered PFO. This was also reflected in unchanged plasma FFA, glucose, and insulin concentrations. In this study we show that plasma FFA availability is closely tied to whole body PFO and that the length of fasting combined with previous exercise are robust stimuli toward increasing plasma FFA concentration, highlighting the importance for preexercise standardization when conducting GXTs measuring substrate oxidation.    NEW &amp; NOTEWORTHY We show that peak fat oxidation is increased in close relationship with plasma free fatty acid availability after combined fasting and repeated incremental exercise tests in healthy highly trained men. Therefore it may be argued that whole body fat oxidation rate measured in most cases after an overnight fast indeed does not represent whole body maximal fat oxidation rate but a whole body peak fat oxidation rate within the context of the preexercise standardization obtained in the study design.

Plasma free fatty acid concentration is closely tied to whole body peak fat oxidation rate during repeated exercise