This study was performed to investigate the effect of water shower spray on rectal temperature (RT), serum levels of corticosterone (CORT), heat shock protein (HSP) 70 and creatine kinase (CK) in broiler chickens road transported at different times of the day. A total of 320 male broiler chickens (35 days of age) were crated and subjected to either water shower spray (WS) for 10 minutes or no water shower (control). The WS and control broilers were then loaded into an open truck and transported either from 0900 h to 1100 h (29.0 o C and 89.0% RH) (AM) or 1900 h to 2100 h (26.0 o C and 94.0% RH) (PM) with an average speed of 80 km/h. The CORT of AM birds were signi cantly reduced by WS, but the treatment had negligible in uence on the PM group. Transportation during AM signi cantly elevated RT, CORT and HSP70 compared to the PM group. Water shower spraying of birds before transit signi cantly reduced RT and HSP70 compared to controls. Neither water shower treatment nor transportation time had a signi cant on CK and mortality rate of broilers. In conclusion, water shower spray is bene cial to alleviate the physiological stress response in road transported broilers under the hot and humid tropical environment.