Objective To observe the changes of plasma metabolites in rats after heat acclimation and following exercise stimulation, so as to screen the specific marker of heat acclimation. Methods A total of 16 SD rats were randomly divided into control and heat acclimation groups (n=8); rats in the heat acclimation group received 2 hour training a day and those in the control group received no training. After 14 days of heat acclimation, rat orbital blood was collected from control and heat acclimation groups. After a rest for 4 days, rat arterial blood samples were collected under anesthesia after exercising in a thermal environment for 30 min in both groups. The changes of metabolites in the plasmas from control and heat acclimation groups were examined by the nuclear magnetic resonance (NMR)using Bruker 600 MHz, and the changes of plasma metabolites were analyzed after heat acclimation and stimulation with exercise in thermal environment in rats. Results Compared with control group,the heat acclimation group had significantly increased plasma unsaturated lipid, glutamate, lipid and dimethylamine(P<0.05),and significantly decreased glucose, myo-inositol, taurine, threonine and trimethylamine N-oxide (TMAO)(P<0.05). After heat stimulation with exercise in thermal environment the plasma TMAO, glutamate and lipid were significantly decreased in the heat acclimation group (P<0.05). Conclusion After heat acclimation and exercise stimulation in thermal environment, plasma metabolites undergo great changes in rats. TMAO may serve as a marker of heat acclimation, which provide evidence for establishing evaluation system for heat acclimation.