Strenuous exercise in traditional polo matches creates enormous stress on horses. Hematological and physiological measures may vary across different field-play positions. This study aimed to investigate the effort intensity and the impact of exertion on hematology and heart rate variability (HRV) in polo ponies with different positions. Thirty-two ponies, divided equally into eight teams, were studied. Each comprises forwards (number 1), midfielders (numbers 2 and 3), and defenders (number 4). Team pairs played the first chukka in four low-goal polo matches. Percent maximum heart rate (%HRmax), indicating ponies' effort intensity, was classified into five zones, including zones 1 (<70%), 2 (70-80%), 3 (80-90%), 4 (90-95%) and 5 (>95%). Hematological and HRV parameters were determined before, immediately after, and at 30-minute intervals for 180 minutes after chukkas; HRV variables were also obtained during warm-up and exercise periods. Results indicated that the number two ponies spent more time in zone 4 (p < 0.05) but less in zone 2 (p < 0.01) than the number four ponies. Cortisol levels increased immediately and 30 minutes afterward (p < 0.0001 for both) and then returned to baseline 60-90 minutes after exertion. Other measures (Hct, Hb, RBC, WBC, neutrophils, and CK enzyme) increased immediately (p < 0.0001 for all) and lasted at least 180 minutes after exertion (p < 0.05-0.0001). HRV decreased during the chukka until approximately 90 minutes afterward (p < 0.05-0.0001). The stress index increased during the chukka and declined to baseline at 60 minutes in number 1-3 ponies but lasted 90 minutes in those at number four. Effort intensity distribution differed among field-play positions. Decreased HRV indicated reduced parasympathetic activity during exercise, extending to 90 minutes after exertion in polo ponies. Defenders seem to experience more stress than those in other positions.