Functional capacity is the physical ability to perform a defined task. In humans it can be evaluated by using exercise testing, for instance sub-maximal exercise tests. Walking tests are widely used variations of submaximal exercise tests and in human medicine are considered to be realistically related to daily physical activity and prognosis in patients with heart failure. The aim of this study was to assess a sub-maximal exercise test in dogs with varying degrees of heart disease. The 6-minute walking test was used to test the functional capacity in healthy dogs and dogs with heart disease (degenerative mitral valve disease). Three groups of untrained owned dogs were compared. Two groups were dogs had mild and moderate degenerative mitral valve disease (ME-1 and ME-2, respectively) and the third group comprised healthy dogs with the same age and size conditions. Both groups of dogs with mitral valve disease walked shorter distances during 6 min (control 448.92 m; ME-1: 406.89 m; ME-2: 350.04 m). The influence of the test on the heart rate, electrocardiography and blood pressure was also evaluated. Although the 6-minute walking test has already been tested in healthy dogs, dogs with chronic pulmonary disease and dogs with artificially induced heart disease, this is the first time that this test has been validated in elderly dogs with naturally acquired heart disease. Thus, we demonstrate here that this test can be used reliably for assessing functional capacity in dogs with heart disease.