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Background/Aims: The effects of muscle cooling on the stiffness of the human gastrocnemius muscle (GAS) were examined in vivo. Methods: The knee joint was passively extended from 90 to 0° (0° = full knee extended position) with a constant ankle angle of 10° dorsiflexed position (0° = the sole of the foot is approximately perpendicular to the anterior margin of the shaft of the tibia) in a control condition (room temperature of 18–23°C) and a cooling condition (muscle temperature decreased by 5.8 ± 1.7°C after cooling using a cold water bath at a temperature of 5–8°C for 60 min). The change in passive Achilles tendon force, muscle fascicle length of GAS and muscle temperature were measured (n = 6) during the motion. Results and Conclusion: GAS stiffness was significantly greater in the cooling condition (20 ± 8 N/mm) than the control condition (18 ± 8 N/mm). There was no cooling effect on the muscle slack length, beyond which passive muscle force arises. The maximum passive Achilles tendon force significantly increased by 19 ± 20% after cooling. These results suggested that cooling increased the passive muscle force due to the increase in the muscle stiffness rather than the shift of the muscle slack length.