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We examined the variability and determinants of the respiratory exchange ratio (RER) at rest and during exercise in 61 trained cyclists. Fasting (10-12 h) RER was measured at rest and during exercise at 25, 50, and 70% of peak power output (W(peak)), during which blood samples were drawn for [lactate] and [free fatty acid] ([FFA]). Before these measurements, training volume, dietary intake and muscle fiber composition, [substrate], and enzyme activities were determined. There was large interindividual variability in resting RER (0.718-0.927) that persisted during exercise of increasing intensity. The major determinants of resting RER included muscle glycogen content, training volume, proportion of type 1 fibers, [FFA] and [lactate], and %dietary fat intake (adjusted r(2) = 0.59, P < 0.001). Except for muscle fiber composition, these variables also predicted RER at 25, 50, and 70% W(peak) to different extents. The key determinant at 25% W(peak) was blood-borne [substrate], at 50% was muscle [substrate] and glycolytic enzyme activities, and at 70% was [lactate]. Resting RER was also a significant determinant of RER at 25 (r = 0.60) and 50% (r = 0.44) W(peak).