This study aimed to assess the variation over time in thermal comfort indices and the behavior of physiological parameters related to thermolysis, blood parameters, and semen in natura of buffalo bulls reared in tropical climate. The study was carried out in an artificial insemination station under a humid tropical climate (Afi according to Köppen). Ten water buffalo bulls (Bubalus bubalis) were used during the 5 months (April to August) of study. The environmental Temperature Humidity Index (THId) and the pen microclimate Temperature Humidity Index (THIp) were calculated. Every 25 days, respiratory rate (RR), heart rate (HR), rectal temperature (RT), and Benezra's thermal comfort index (BTCI) were assessed in the morning and in the afternoon. A blood assay was performed every month, while semen was collected weekly. THIp did not vary over the months (P > 0.05) and was higher in the afternoon than in the morning (77.7 ± 2.6 versus 81.8 ± 2.1, P < 0.05). RR, HR, and BTCI significantly increased over the months and were different between the periods of the day (P > 0.05) but within the physiological limits. RT varied between the periods of the day and decreased over the months, being the lowest in August (37.8 ± 0.7 °C), time-impacted hematocrit, mean corpuscular volume, hemoglobin levels, and spermatic gross motility and vigor (P < 0.05). Thus, buffalo bulls reared under a humid tropical climate may have variations in thermal comfort during the hotter periods but are able to efficiently activate thermoregulatory mechanisms and maintain homeothermy, hence preserving their physiological and seminal parameters at normal levels.