Objective: The aim of the present study was to investigate the effects of repeated thermal conditioning (RTC) at an early age on physiological and behavioral responses in chicks. Methods: Birds were assigned to one of the four treatments in which the RTC was exposure to 40 °C for 15 min daily. The treatments were 1) no thermal conditioning (control); 2) early exposure group (EE; RTC from 2 to 4 days of age); 3) later exposure group (LE; RTC from 5 to 7 days of age); or 4) both early and later exposure (BE; RTC from 2 to 7 days of age). All groups of chicks were challenged with high ambient temperature (40 °C for 15 min) at two weeks of age. Results: During heat challenge, initiation times of dissipation behaviors (panting and wing-drooping) were measured. Rectal temperature and respiration rate were measured after and before heat challenge. Hypothalamic samples and blood were collected at the end of heat challenges. Initiation times of dissipation behaviors and rectal temperature were not affected by the treatments. Increases in respiration rate in response to heat challenge were suppressed by early RTC treatment. There was no clear pattern of glucose levels in relation to thermal conditioning, whereas plasma corticosterone levels were decreased by early treatment (EE and BE groups). Hypothalamic thyrotropin releasing hormone gene expression was suppressed by early and later thermal conditioning and suppressed further by both early and later exposure. Neuropeptide Y gene expression in the BE group was lower than in the other groups, with a similar trend for corticotropin releasing hormone expression. Conclusion: Our results suggest that the effect of repeated thermal conditioning on the central thermoregulatory system depends on the number of times that chicks experienced conditioning. In addition, repeated thermal conditioning has greater effects on the acquisition of thermotolerance when conditioning occurs in chicks of two to four days of age in comparison with chicks of five to seven days of age.