Skeletal muscles are essential to the gender-specific characteristics of human movements. Sonomyography, a new signal for quantifying muscle activation, is of great benefit to understand muscle function through monitoring the real-time muscle architectural changes. The purpose of this pilot study was to investigate gender differences in the architectural changes of gastronomies muscle and tendon by using sonomyography during performing two-legged calf raising exercises. A motion analysis system was developed to extract sonomyography from ultrasound images together with kinematic and kinetic measurements. Tiny fascicle length changes among seven male subjects were observed at the initial part of calf raising, whereas the fascicle of seven female subjects shortened immediately. This result suggested that men would generate higher mechanical power output of plantar flexors to regulate their heavier body mass. In addition, the larger regression coefficient between the fascicle length and muscle force for the male subjects implied that higher muscle stiffness for the men was required in demand of maintaining their heavier body economically. The findings from the current study suggested that the body mass might play a factor in the gender difference in structural changes of muscle and tendon during motion. The sonomyography may provide valuable information in the understanding of the gender difference in human movements.