This study aimed to explain the slow component ( ) and muscle fatigue during cycling at different intensities. The muscle fatigue of 16 participants was determined through maximal isokinetic effort lasting 3 s during constant work rate bouts of moderate (MOD), heavy (HVY) and very heavy intensity (VHI) exercise. Breath-by-breath , near-infrared spectroscopy signals and EMG activity were analysed (thigh muscles). was higher during VHI exercise (∼70% vs. ∼28% of reserve in HVY). The deoxygenated haemoglobin final value during VHI exercise was higher than during HVY and MOD exercise (∼90% of HHb physiological normalization, vs. ∼82% HVY and ∼45% MOD). The muscle fatigue was greater after VHI exercise (∼22% vs. HVY ∼5%). There was no muscle fatigue after MOD exercise. The greatest magnitude of muscle fatigue occurred within 2 min (VHI ∼17%; HVY ∼9%), after which it stabilized. No significant relationship between and muscle force production was observed. The τ of muscle was significantly related (R2 = 0.47) with torque decrease for VHI. Type I and II muscle fibre recruitment mainly in the rectus femoris moderately explained the muscle fatigue (R2 = 0.30 and 0.31, respectively) and the (R2 = 0.39 and 0.27, respectively). The is also partially explained by blood lactate accumulation (R2 = 0.42). In conclusion muscle fatigue and O2 cost seem to share the same physiological cause linked with a decrease in the muscle and a change in lactate accumulation. Muscle fatigue and are associated with muscle activation heterogeneity and metabolism of different muscles activated during cycling.