In clinical settings, no method has been established to examine the fatigue of a latissimus dorsi muscle (LDM) preconditioned for cardiomyoplasty. We examined the feasibility of measuring muscle stiffness (tactile stiffness) to evaluate muscle fatigue in situ using our tactile sensor. We stimulated canine LDM with burst pacing and monitored both stiffness and tension to determine their relationship. In both dissected LDM and LDM in situ, the decrements of these parameters during burst pacing were compared between preconditioned and unconditioned LDM. In measurement in situ, the sensor probe was placed on the LDM through a small incision. Strong statistical correlation was shown between stiffness and tension (r = 0.935). In decrements of stiffness in situ, there were statistically significant differences between preconditioned and unconditioned LDM. Our tactile sensor system can provide an efficient method for evaluating fatigue of muscles in situ without measuring muscle tension.