This study examined whether different neurotransmitters are presented at exercise fatigue in different temperature and relative humidity (RH) environments in athletes. Eight trained male athletes performed exercise in five different environmental conditions: 21°C/20% RH (Normal); 33°C/20 % RH (Hot 20%), 33°C/40% RH (Hot 40%), 33°C/60% RH (Hot 60%), and 33°C/80% RH (Hot 80%). Exercise group performed VO2max test in five conditions. Blood samples were taken pre‐ and post‐exercise and analyzed for noradrenaline (NA), adrenaline (Ad), dopamine (DA), serontonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), prolactin (PRL). Compare to Normal condition, Hot 20%, Hot 40% and Hot 80% have lower VO2max (P < 0.05). A comparison between the means indicated that in Hot 20%, Hot 40%, Hot 60% and Hot 80% conditions the RPEmax was higher than the Normal condition (P < 0.01 or P < 0.05). There was a significant effect for time in NE (P < 0.0001), PRL (P < 0.0001), 5‐HT (P = 0.002), 5‐HIAA (P = 0.029), DA (P = 0.016) during exercise in different conditions. However, Ad did not show any significant effect between pre and post exercise (P >; 0.05). In different humidity and heat environments, exercise fatigue is determined by the collaboration of the different neurotransmitter systems, with the most important role possibly being for the NE, 5‐HT and DA.This work was supported by Grants from Ministry of Science and Technology of the People's Republic of China (2010–05).