The purpose of this study was to describe the effect of increasing workload on individual thigh muscle activation during a 20 minute incremental cycling test. Intramuscular electromyographic signals were recorded from the knee extensors rectus femoris, vastus lateralis, vastus medialis and vastus intermedius and the knee flexors semimembranosus, semitendinosus, and the short and long heads of the biceps femoris during increasing workloads. Mean activation levels were compared over the whole pedaling cycle and the crank angles at which onset and offset of activation and peak activity occurred were identified for each muscle. These data were compared between three workloads. EMG activation level significantly increased (p<0.05) with increasing workload in the rectus femoris, vastus medialis, vastus lateralis, vastus intermedius, biceps femoris long head, semitendinosus and semimembranosus but not in the biceps femoris short head. A significant change in activation timing was found for the rectus femoris, vastus lateralis, vastus medialis and semitendinosus. Of the knee flexors only the short head of the biceps femoris had its peak activity during the upstroke phase at the two highest workloads indicating a unique contribution to knee flexion.