Thermal conditioning of chicks results in improvements in performance and thermotolerance at marketing age. Conditioning has been found to be a sensitive process, dependent on age and the temperature used. The objective of this study was to assess the optimal timing and temperature for the conditioning process. Six separate trials were conducted on male broiler chickens: the first two aimed to find the optimal age for thermal conditioning (1 to 5 days of age); the other four evaluated the optimal thermal conditioning temperature between 36 and 40.5 C. At 42 day of age chickens were thermally challenged to evaluate their ability to cope with acute heat stress. The highest body weight was achieved when thermal conditioning had been applied at the age of 3 days, and it coincided with low feed intake and higher to significantly higher feed efficiency. These treated chickens showed relatively lower mortality rate under thermal challenge and lower to significantly lower Ttiiodothyronine (T3) concentration in Trial 2. Chicks that had been thermally conditioned at ambient temperatures (Ta) of 36 and 37.5 C at the age of 3 days demonstrated the best performance characteristics and the ability to reduce T3 concentration to the lowest levels during thermal challenge. It can be suggested, therefore, that a Ta between 36.0 and 37.5 C, applied at 3 days of age is optimum for thermal conditioning of broiler chickens.