This study aimed to verify the effects of marcha exercise on clinical, hematological, and biochemical variables in Mangalarga Marchador horses. Twelve Mangalarga Marchador horses were used, six females, and six males. Each animal was submitted to marcha exercise lasting 40 minutes on hard ground, a training regimen to which these animals were already conditioned. The animals were assessed before exercise (M0) and immediately after (M1). Physical examinations were carried out and blood samples were collected via jugular venipuncture to obtain blood, plasma, and serum for hematological and biochemical profiles. The marcha training caused changes in the values of all clinical and laboratory variables evaluated. The physical examinations revealed increases in cardiac and respiratory rates, as well as rectal temperatures. An increase was observed in erythrocyte, hemoglobin, hematocrit, and leukocyte values. The serum concentrations of electrolytes such as sodium, potassium, chloride, magnesium, and ionic calcium decreased. An increase in strong ion difference (SID) was observed, likely due to the decrease in chloride concentration causing a mild metabolic alkalosis. There was an increase in the plasma concentrations of lactate, glucose, creatine phosphokinase (CK), total protein, and fibrinogen. In conclusion, the marcha causes small changes in the clinical, hematological, and biochemical profiles of horses previously conditioned to this exercise.