Muscle hardness increases as the contractile level increases. This increase is caused by changes in structure of the muscle fiber and blood flow; however, the mechanism of increasing hardness has not been clearly demonstrated. The objective of this study was to investigate the relationship between isolated frog muscle tension and hardness. Gastrocnemius muscles were mounted horizontally in a chamber. The femur was fixed, and the Achilles tendon was attached to a stretching device. The muscle tension and hardness were measured during various muscle stretches and with and without electrical stimulation. We applied two protocols. In the first, the muscle was stimulated and then stretched, whereas, in the second, it was stretched and then stimulated. The muscle hardness was proportional to the muscle tension at each amount of stretching in both protocols. There were no significant differences between protocols 1 and 2, although the stretch enhancement of the muscle force was expected in protocol 1. In our experiments, the muscle length corresponds to the ascending limb of the length-tension curves of a sarcomere. The results of this study suggest that the relationship between muscle tension and hardness was not affected by the stretch enhancement in the ascending limb of the length-tension curve. The slope of the regression line between the muscle tension and hardness decreased as the amount of the stretch increased. The decrease of the slope might be caused by structural changes in the filaments.