Background: Indian Kathiawari horses are well-known as an endurance breed in the Indian subcontinent, yet not much research has been undertaken on their exercise physiology. An evaluation of the exercise physiology of Kathiawari horses could significantly contribute to the further development of the breed and for the improvement of its healthcare. Objectives: This study aimed to investigate the changes in cardiac biomarkers and echocardiographic indices before and after exercise in Indian Kathiawari horses. Methods: Eighteen working Kathiawari horses age ranging from 2 years to 12 years, consisting of 8 mares and 10 stallions were selected for this study. Horses were subjected to exercise for a period of ± 40 mins. Serum samples were collected before and after exercise for evaluation of changes in cardiac biomarkers concentration. Electrocardiographic and echocardiographic evaluations were also done for same horses before and after exercise. Results: CTnI, CTnT, CK-MB and NT-proBNP after exercise showed highly significant increase when compared to before exercise. Significant increase in left ventricular dimensions and systolic function such as LVIDd, LVIDs, EDV, ESV, SV, Ao, LA, EF and FS after exercise when compared to before exercise. Post exercise doppler echocardiographic showed increase in number of aortic regurgitation compared to pre exercise. Significant changes were seen in P and T wave morphology and significant decrease in the duration of P wave, P-Q interval, QT interval and R-R intervals immediately after the exercise. Significant elevation was noticed in P wave amplitude immediately after the exercise. Conclusion: In Indian Kathiawari horses the concentration of cardiac biomarkers are increased after 3 hour of exercise. Increased left ventricular dimensions and systolic function was observed in horses after exercise. This study contributes to establishing the reference ranges of cardiac biomarkers in Kathiawari horses after exercise.