The aim of the current study was to evaluate the effect of different thermal manipulation (TM) protocols during embryogenesis on thermotolerance acquisition parameters during subsequent thermal challenge (TC) at posthatch day 28. A total of 1500 fertile chicken eggs were divided randomly into five treatments (300 eggs each): control was maintained at 37.8 °C and 56 % relative humidity (RH) whereas, TM1, TM2, TM3 and TM4 were subjected to 38.5, 39, 39.5 and 40 °C for 18 h and 65 % RH daily during embryonic days ED 12–18. Hatched chicks from each treatment group allocated randomly into two sub-treatment groups (thermo-neutral, naïve (TN) and thermal challenge (TC). At day 28 of age, chicks subjected to TC by adjusting room temperature to 42 °C for 6 h while naïve chicks kept under regular conditions (22 ± 1 °C and 50–60 % RH). Chick’s response to TC evaluated by determination of plasma T3, T4, corticosterone, total proteins, albumin, selected enzymes and some electrolytes at the beginning (0 h) and after 1, 3 and 5 h of TC in TM and TN chicks. Furthermore, pectoral and thigh muscles mRNA expression of Atrogin-1, CK, avUCP, DIO3, DIO2 were evaluated in TC and TN sub-treatment groups.