It is uncertain whether fatigue and workload would affect muscle recruitment during cycling. To infer on changes in priority for recruitment of motor units, we assessed the combined effects of fatigue and workload during an incremental cycling test to exhaustion on frequency components of lower limbs muscle activation. Competitive cyclists performed an incremental maximal cycling test while muscle activations were recorded from the right and left vastus lateralis, biceps femoris and gastrocnemius medialis. Muscle activation signals were assessed using frequency band analyses and decomposed into overall, high and low frequency bands. Combined effects from workload/fatigue were assessed using t tests and Cohen¿s effect sizes (ES). There were increases in the overall muscle activation due to increased workload/fatigue for biceps femoris (40% vs. 90%, p<0.01 and ES = 1.85) and vastus lateralis (40% vs. 70%, p=0.01 and ES = 0.96, and 40% vs. 90%, p<0.01 and ES = 2.03, and 70% vs. 90%, p<0.01 and ES = 0.96), but not for gastrocnemius medialis. There was also greater contribution from low frequency component for biceps femoris (40% vs. 90%, p = 0.01 and ES = 1.12). Similar workload/fatigue effects have been observed between lower limbs. In conclusion, incremental cycling test lead to an increase in activation of main knee joint flexors and extensors but not in plantar flexors during cycling. Biceps femoris changes its recruitment profile due to increases in low frequency content.