Parasympathetic tone activity (PTA) and its association with behavior in conscious versus anesthesia in horses are unknown. To study PTA, heart rate (HR), minimum alveolar concentration (MAC), and mean arterial pressure (MAP) during castration under anesthesia. To study PTA, HR, Horse Grimace Scale (HGS), locomotor activity (LA), and behavior in conscious horses before and after castration. Pilot study including seven healthy adult horses. After habituation (days 1, 2, and 3), PTAm, HR, HGS, LA, and behavior were investigated during pre-surgery (days 4-, 5-, and one-hour pre-anesthesia), and post-surgery (6 h post-surgery and days 7 and 8) periods. PTAm, HR, MAP, and MAC were recorded during castration. PTAm increased significantly during anesthesia (P < 0.001), with sequential increase as time passed (P < 0.001): 15 min = 27.4% (62.6, CI 57.3-67.8), 20 min = 38.2% (70, CI 55.7-84.3), 25 min = 59.6% (87.9, CI 76.3-99.4), 30 min = 55.6% (84.1, CI 76.8-91.5). MAP increased significantly after emasculation (P < 0.003) at 1 min (91 mmHg, CI 77.3-104.7) and 5 min (81 mmHg, CI 67.3-94.7). An increase HGS (P < 0.05), PTAm (Δ16%, P < 0.001), head up latency (P = 0.042), and frequency of limb retraction (P = 0.042), while a decrease in LA (P = 0.004), appetite (P = 0.043), and water consumption (P < 0.05) in days post-surgery were observed. Modulation of vagal tone occurs in response to castration under anesthesia and preservation of homeostasis days post-surgery.