Plasma free fatty acids (FFA) are a major contributor to whole body fat oxidation during exercise. However, to what extent, 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 peak fat oxidation rate in a dose dependent manner. 10 healthy young (31±6 years) (mean ± SD) well-trained (V̇O_2max: 65.9±6.1 ml/min/kg) men performed 4 graded exercise tests (GXT) on one day. The GXT were interspersed by 4 hours of bedrest. This was conducted either in fasted state, or with the consumption of a standardized carbohydrate-rich meal 31/2 hours before each GXT. Fasting and previous GXT resulted in a gradual increase in PFO from 0.63±0.18g/min after an overnight fast (10 hours) to 0.93±0.17 g/min after app. 22 hours of fasting and three previous GTX. This increase in PFO coincided with an increase in plasma FFA concentrations (r²=0.73, p<0.0001). Ingestion of a carbohydrate-rich meal 31/2 hour before each GXT resulted in unaltered PFO rates. 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 peak fat oxidation rate and that the length of fasting and previous exercise are strong stimuli towards increasing plasma FFA concentration, highlighting the importance for pre-exercise standardisation when conducting graded exercise tests measuring substrate oxidation.