The aim of the present study was to evaluate the effect of the induced heat stress on physiological response and serum biochemical parameters involving glucose, cholesterol, blood urea nitrogen (BUN), non-esterified fatty acids (NEFA), and cortisol in Holstein calves. Ten calves were kept in a climate controlled room (air temperature 37℃ and 90% humidity from 09:00 to 19:00) for three days. Those animals were given a one-day adaptation period. During the treatment period, we measured the skin temperature six times. Following the treatment periods, blood samples were collected before the experiment began (09:00) and at the end of the stress period (19:00). To aid analysis of the biochemical parameters, also we monitored the rectal temperature. The results, exhibited that both rectal and skin temperature showed increase in the heat stress-induced animals as compared with un-stressed animals. Moreover, we noticed that the levels of BUN and NEFA increased in the blood serum of heat stress induced animals when compared with un-stressed ones. From these results, we concluded that the physiological and biochemical changes in the calves were induced by heat stress. Hence, the present study findings could be employed as base line data for development of stress reduction techniques in the dairy industry.