The purpose of this study is to establish a quantitative method that can evaluate the motor function of human lower limbs in dynamic situations. As an index of motor function, we focus on the mechanical impedance characteristics of the lower limbs. By controlling these characteristics, humans can improve motion stability and adapt their movement to disturbances in the environment. In this paper, we propose a method that can estimate the impedance characteristics of the lower limbs of a human subject applying torque perturbations during a pedaling exercise. To improve the accuracy of the proposed estimation method, the effects of inertia and gravity in the measurement data were considered by representing the lower limbs by a multi-link model. Experiments were performed in two participants to estimate the stiffness and viscosity at different phases of the periodic movement. The distributions of these parameters showed symmetric properties for both legs in a pedaling cycle-a tendency that was observed in both participants. This agreement suggests that our method could extract a component of human motor control strategy that adjust mechanical impedances during pedaling exercises.