The present study was conducted to determine the effect of increasing temperature humidity index (THI) on lactating Murrah buffalo. The study was conducted on six lactating buffaloes which were offered the same basal diet and were blocked by days in milk, milk yield and parity. The study was conducted during May, June and July with average THI of 79.88, 80.57 and 85.36, respectively. Milk yield was recorded twice a day at 06:00 and 18:00 h and physiological parameters were recorded and blood was sampled on 30th day of each month at 14:30 h. The results showed a significant decrease (P<0.05) in milk yield in buffaloes only at a THI of 85.36 as compared to THI up to 80.57. The rectal temperature (RT) did not change (P=0.580) with increasing THI up to 85.36, however, the respiration rate (RR) and pulse rate (PR) changed (P<0.05) progressively with increasing THI. Leucocytopnoea, lymphocytopnoea and neutrophilia; and alteration in serum urea, alkaline phosphatase activity, reactive oxygen species and phosphorus concentration were observed only at a THI of 85.36 as compared to THI up to 80.57. Similarly, significant (P<0.05) alteration in serum T4, cortisol and prolactin levels were observed at a THI of 85.36 as compared to THI up to 80.57. It can be concluded that lactating buffaloes experienced mild heat stress up to a THI of 80.57 (May and June) and a moderate type of heat stress at a THI 85.36 (July).