We examined muscle stiffness at various pedaling rates under conditions of constant power output. Eight healthy young participants pedaled a cycle ergometer at a power output of 47 W. The combinations of pedaling rate and workload were 40 revolutions per min (rpm) and 11.7 N, 60 rpm and 7.8 N, and 80 rpm and 5.9 N, respectively. One electrical stimulus per two pedal rotations was applied to the vastus lateralis muscle at a crank angle of 30° in the down phase. Mechanomyograms (MMGs) were measured using a capacitor microphone, and the evoked MMG was extracted. An evoked MMG system was identified, and the coefficients of the denominator of the transfer function were used to estimate stiffness and viscous coefficient of the muscle. Muscle stiffness was 236–705 Nm⁻¹, and was proportional to the pedaling rate when power output was held constant, while the viscous coefficient did not change from approximately 15 Nm⁻¹s. In conclusion, our findings demonstrate that stiffness of the vastus lateralis muscle increases with increasing pedaling rate under conditions of constant power output, while the viscous coefficient does not change.