The purpose of this study was to investigate the effects of temperature change on serum FFA, serum glucose concentration and energy expenditure during prolonged exercise. All seven healthy males were asked to do exercise at 60% VO2max in the chamber and experimental temperature within the chamber were adjusted to three condition : normal temperature(NT:20±0.5℃)/ low temperature(LT:5±0.5℃), high temperature(HT:35±0.5℃) and 50±3% humidity condition. Each experimental exercise were monitored and analyzed the change of serum FFA, serum glucose and energy expenditure. Serum FFA concentration showed a significant(p＜.05) higher value in LT at exercise 30min, post exercise 15min than NT and HI, serum glucose concentration showed a significant(p＜.05) higher value in HT at post exercise, recovery 15min, recovery 30min than NT and LT. In summary, LT condition showed energy supply depend on serum FFA, HT condition showed energy supply depend on serum glucose during prolonged exercise.