Long-term endurance exercise could increase activity of parasympathetic nervous and decrease activity of sympathetic nervous at rest. However, previous studies all focused on the effect of endurance training on heart rate variability (HRV) for athletes or sedentary subjects. In Taiwan, elementary school teachers teaching and processing the children's and administrative problems always stand and walk. They will sit down only when they review and correct the students' home work. Thus, the goal of this study was to elucidate the beneficial effect of moderate intensity exercise on relieving mental load of elementary school teachers. There were 20 participants in the exercise group and another 20 participants in the nonexercise group. The exercising teachers performed 12 weeks of moderate intensity exercise training for an average of 30 minutes per day, 3 times per week. HRV was measured before and after the 4th, 6th, and 12th weeks. The time and frequency domain parameters of HRV all had significant increases between the beginning and after 12 weeks of training. However, the time and frequency domain parameters of HRV in the nonexercise group had significant decreases between the beginning and after 12 weeks of training. The long-term moderate exercises can relieve mental load of elementary school teachers. Moreover, age was the considerable factor affecting HRV in this study.