The article presents the results of research on the activity of cardiac specific enzymes and the concentration of lactate in the blood of sport horses for myocardial dystrophy of physical activity. Materials for researches were horses, used in the classical forms of equestrian sport of Ukrainian riding (n = 20), Hanoverian (n = 15) and Westphalian (n = 15) breeds. The average age of horses was 8.4 ± 0.71 years (3.5–16.0 g.), weight – 479.4 ± 8.54 kg (350–605 kg). Studies were conducted immediately before and immediately after exercise. The duration of regular training of average intensity was 1 hour: step 5 min; roaring lynx 10 minutes; step 5 min; training lynx 10 minutes; step 10 min; gallop with a transition to a step of 10 minutes; step 10 min. Blood samples from horses were taken from a jugular vein in a test tube without an anticoagulant (10 ml; Vacutest, Italy) and an anticoagulant (EDTA-K, 2.0 ml; Sarstedt, Germany). Diseases of the heart muscle are accompanied by the release of substances released from damaged cardiomyocytes, in particular, creatine kinase (CK), lactate dehydrogenase (LDH), myoglobin. It has been established that the key to the pathogenesis of myocardial dystrophy in sport horses is the discrepancy between costs and energy recovery in the functioning structures of the heart muscle due to excessive cardiac load and significant increase in energy costs, as well as a disturbance in the balance of the electrolytes. Hyperlactatemia that occurs in horses during exercise causes changes in the permeability of cardiomyocytes and exit enzymes in the blood and can play a key role in the pathogenesis of myocardial dystrophy. Іn sporting horses, after activity in blood serum, activity of AST and LDH increases in blood serum, a tendency to increase activity of creatine kinase (CК) is observed. The cardiac isoenzyme CK (CK‒MB) was detected by a specific marker of myocardial dystrophy in sport horses, since its activity was likely to increase in the blood of all experimental groups of animals. A less specific marker of myocardial damage was the activity of hydroxybutyrate dehydrogenase (LDH‒1) in the blood, since the probable difference was established only in the Westphalian horses. Investigating the activity of AST, CK and CK-MB in horses can serve for differential diagnosis of rhabdomyolysis syndrome and damage to heart muscle cells, in particular for myocardial dystrophy.