Differences in the response of OmniGen-AF (OG) supplemented dairy cows to a corticotropin releasing hormone (CRH) and vasopressin (VP) or an adrenocorticotropic hormone (ACTH) challenge when housed at different temperature-humidity indices (THI) were studied. Holstein cows (n = 12; 162 ± 1 DIM) were balanced by milk yield, BW and DIM into 1 of 2 trts: 1) OminGen-AF, supplemented with OG at 4.54 g/kg BW for 70d; or 2) Control (CON), no supplement. Cows were moved to individual tie stalls in 1 of 2 temperature controlled chambers on d51 and fitted with indwelling RT devices and jugular catheters on d52. Initially THI was cycling at thermoneutrality (TN; THI < 72 for 24 h/d) for 7d, followed by HS (THI > 72 for 12h/d) for 10d. Cows were challenged with CRH (0.3 µg/kg) and VP (1 µg/kg) at 1000h on d4 of TN and d1 of HS, and with ACTH (0.1 IU/kg) at 1000h on d5 of TN and d2 of HS. Blood samples were collected from −2 to 8h at 30-min intervals relative to each challenge and analyzed for leukocyte profiles. There was a THI × time interaction (P ≤ 0.01) for RT such that RT was greater during HS than TN (2 to 9h, and at 11h for CRHVP and 4 to 9h and 11 to 13h for ACTH). Also, RT was greater (P ≤ 0.02) in OG than CON cows regardless of challenge. Total white blood cells (WBC) and neutrophils (NT) increased (P < 0.01) in response to CRHVP and ACTH. There was a trt × THI interaction (P < 0.01) for WBC during both challenges. Specifically, WBC were decreased in CON during HS compared with TN in response to CRHVP, while WBC were greatest in CON during TN and least in OG during HS in response to ACTH. There was a trt x THI interaction (P = 0.02) for NT in response to CRHVP challenge; NT decreased in CON cows during HS compared with TN. Also, NT were decreased during HS (P < 0.01) compared with TN and decreased in OG compared with CON (P < 0.01) following ACTH. Lymphocytes were decreased during HS compared with TN (P < 0.01) and in OG compared with CON cows (P < 0.01) regardless of challenge. These data suggest supplementing cows with OG can reduce the negative effects of HS on leukocyte profiles following activation of the stress axis.