Good horse performance depends on a genetic predisposition, training, health status, and optimal values of hematological and biochemical blood parameters. This research aimed to monitor 20 hematological and 15 biochemical blood parameters during different types of physical activity in clinically healthy 20 sports and 20 working horses with an average age of 8.33 years. Sport male horses had higher MCV, MCH, number, and percentage of neutrophils, eosinophils, creatinine, and bilirubin, while mares had higher values of RDW, lymphocytes, monocytes, basophils, MPV, glucose, BUN-to-creatinine ratio. Physical activity in draft horses led to a statistically significant increase in the number of leukocytes and neutrophils and significant hypocalcemia, while in sport horses, there was a significant increase in RBC, HCT, and HGB and significant hypoglycemia after rest. Significantly higher values of neutrophils and creatinine were recorded in male draft horses, while in mares, there were higher values of lymphocytes and amylase. This study proved that sport horses have better aerobic capacity and performance, are better adapted to the type and intensity of physical activity, and are kept in better conditions than draft horses. The different types of physical activity and other physiological factors can significantly change hematological and biochemical parameters in the draft and sports horses. Monitoring of hematological and biochemical blood parameters during different types of exercise is essential to assessing horses' performance, welfare status, and health status.