This study aimed to clarify the acute effects of static stretching (SS) and cyclic stretching (CS) on muscle stiffness and hardness of the medial gastrocnemius muscle (MG) by using ultrasonography, range of motion (ROM) of the ankle joint and ankle plantar flexor. Twenty healthy men participated in this study. Participants were randomly assigned to SS, CS and control conditions. Each session consisted of a standard 5-minute cycle warm-up, accompanied by one of the subsequent conditions in another day: (a) 2 minutes static stretching, (b) 2 minutes cyclic stretching, (c) control. Maximum ankle dorsiflexion range of motion (ROM max) and normalized peak torque (NPT) of ankle plantar flexor were measured in the pre- and post-stretching. To assess muscle stiffness, muscle-tendon junction (MTJ) displacement (the length changes in tendon and muscle) and MTJ angle (the angle made by the tendon of insertion and muscle fascicle) of MG were measured using ultrasonography at an ankle dorsiflexion angle of –10°, 0°, 10° and 20° before and after SS and CS for 2 minutes in the pre- and post-stretching. MG hardness was measured using ultrasound real-time tissue elastography (RTE). The results of this study indicate a significant effect of SS for ROM maximum, MTJ angle (0°, 10°, 20°) and RTE (10°, 20°) compared with CS (p < 0.05). There were no significant differences in MTJ displacement between SS and CS. CS was associated with significantly higher NPT values than SS. This study suggests that SS of 2 minutes’ hold duration significantly affected muscle stiffness and hardness compared with CS. In addition, CS may contribute to the elongation of muscle tissue and increased muscle strength.