This investigation was undertaken to clarify the in vitro effect of the various stimulations, such as exercise (E), insulin (I) direct electrical stimulation (EST) and the combinations of the above, on the glucose incorporation intro glycogen molecules (glycogen synthesis) of the normal slow (soleus) and fast twitch (plantaris) muscles, and the different responses of slow and fast twitch muscles to persistent overloads causing compensatory muscle hypertrophy. In resting state, slow twitch muscle has greater capacity for glycogen synthesis than fast twitch muscle, and responses of different muscle to various stimuli were differ as follows: In slow twitch muscle, the glycogen synthesis was increased by insulin, and electrical stimulation but not increased by exercise; exercise increased insulin sensitivity and the effect of electrical stimulation. Whereas the glycogen synthesis in fast twitch muscle was increased only by the stimuli combined with E and EST, and E, I, and EST. As the result of removal of synergistic muscle, both muscles were hypertrophied, and the degree of hypertrophy in response to persistent overload was higher in fast twitch muscle (182%) than slow twitch muscle (151%). In hypertrophied muscles, glycogen synthesis of soleus in any groups was lower than that of the control, but similar in plantaris. In conclusions, there were marked heterogeneity in different muscle fiber in the effects of exercise and insulin addition and electrical stimulation on muscle glycogen synthesis, and fast twitch muscle may be adapted more easily to that kind of persistent overload than slow twitch muscle.