Eco-driving is a driving mode through driver behaviors mainly about accelerator pedal operations, including smooth acceleration at start, preservation of steady speed, and active uses of engine brake, and proposed for reduction of vehicle CO2 discharges. In this paper, driving experiments by subjects were conducted in the modes of normal driving, eco-driving, and eco-driving with eco-indicator in virtue of a universal driving simulator (DS). In the experiments, signals of surface electromyography (sEMG) of leg muscles of driver were measured to clarify the degree of muscle fatigue, and effect of mental workload of driver was also comparatively analyzed by a subjectivity evaluation of NASA-TLX method in the different driving modes. The studies suggested that the fuel economies can be improved by the eco-driving mode, which requires more muscular activities of leg for accelerator pedal operations than the normal driving. However, the muscular activities of leg can be decelerated by using the eco-indicator during eco-driving. Moreover, the evaluation results about mental workload shown that drivers always tend to feel mental workload for achieving target values of eco-indicator during the eco-driving.