This study analyzed the effect of time of trot on hematology and blood chemistry values of the Jeju Pony crossbreed horses that are commonly used for riding (14.1±1.4 years old, Gelding). A total of 28 parameters including vital signs as well as stress hormones such as cortisol and lactic acid levels were examined as the time of the trot exercise progressed. Vital signs such as heart rate (38.0→81.0 times/min) and respiratory rate (11.7→35.7 times/min) increased significantly within 30 minutes of exercise. However, difference in the body temperature was not observed before and after exercise. The hematology including white blood cell count (8.03→9.52×103 cells/μL), red blood cell count (5.94×103→7.23–7.32×103 cells/μL), hemoglobin levels (11.82→14.65–14.78 g/dL), and hematocrit levels (25.04→30.27%) significantly increased 30 minutes after the start of the exercise (p<0.05). The blood chemistry value of albumin (3.25→3.47 g/dL) (p<0.05) only showed a significant increase after the exercise. However, the other blood chemistry levels such as, Na+, K+, Ca2+, total CO2, creatine kinase, glucose, blood urea nitrogen, creatinine, aspartate transaminase, total bilirubin, gamma–glutamyl transpeptidase, and total plasma protein did not change. Also, cortisol and lactic acid levels did not show significant difference. The middle-aged Jeju pony crossbreed horses were not stressed by the 30-minute exercise; therefore, it can be concluded that there is no problem regarding the safety of both the rider and the animal.