The purpose of this study was to investigate the effect of intensive eccentric exercise on hamstring muscles by using magnetic resonance imaging (MRI) and to elucidate the relationships between the changes in the electromyographic (EMG) parameters and in the transverse relaxation time (T2) of the hamstring muscles. Seven male volunteers performed eccentric knee flexion exercise, and the EMG activity of the hamstring muscles was simultaneously measured. Before and immediately after the exercise, the maximum isometric knee flexion torque was measured and MR images of the hamstring muscles were obtained. For all hamstring muscles, the EMG activity of the fifth set was significantly lower than that of the first set. For each subject, a significant correlation was detected between the percentage change in the value of the post-exercise T2 value and those of EMG signals during the exercise only for the semitendinosus (ST) muscle and not for the biceps femoris (BF) and the semimembranosus (SM) muscles. These results suggested that the EMG-activity reductions in the BF, ST, and SM muscles were due to neuromuscular fatigue, and moreover the reduction in the ST muscle was due to a failure in the E-C coupling, which was caused by excessive muscle-fiber damage.