Exercise intolerance is the first symptom of heart disease. Yet an objective and standardised method in canine cardiology to assess exercise capacity in a clinical setting is lacking. In contrast, exercise testing is a powerful diagnostic tool in humans, providing valuable information on prognosis and impact of therapeutic intervention. To investigate whether an exercise test reveals differences between dogs with early stage mitral regurgitation (MR) and dogs without cardiac disease, 12 healthy beagles (healthy group, HG) and 12 dogs with presymptomatic MR (CHIEF B1 / B2, patient group, PG) underwent a six-stage submaximal exercise test (ET) on a motorised treadmill. They trotted in their individual comfort speed for three minutes per stage, first without incline, afterwards increasing it by 4% for every subsequent stage. Blood samples were taken at rest and during two 3-minute breaks in the course of the test. Further samples were taken after the completion of the exercise test and again after a 3-hour recovery period. Measured parameters included heart rate, lactate and the cardiac biomarkers N-terminal pro-B-Type natriuretic peptide and cardiac Troponin I. The test was performed again under the same conditions in the same dogs three weeks after the first trial to evaluate individual repeatability. Cardiac biomarkers increased significantly in both HG and PG in the course of the test. The increase was more pronounced in CHIEF B1 / B2 dogs than in the HG. N-terminal pro-B-Type natriuretic peptide increased from 435 ± 195 to 523 ± 239 pmol/L (HG) and from 690 to 815 pmol/L (PG). cTnI increased from 0.020 to 0.024 ng/mL (HG) and from 0.06 to 0.08 ng/ml (PG). The present study provides a method to assess exercise-induced changes in cardiac biomarkers under clinical conditions. The increase of NT-proBNP and cTnI is more pronounced in dogs with early-stage MR than in healthy dogs. Results indicate that measuring the parameters before and after exercise is adequate and taking blood samples between the different stages of the ET does not provide additional information. Also, stress echocardiography was inconclusive. It can be concluded that exercise testing, especially in combination with measuring cardiac biomarkers, could be a helpful diagnostic tool in canine cardiology.