The objectives of this study were to determine the prevalence of sub-clinical diseases in poorly-performing Standardbred horses, compare their physiological response to exercise with control horses, and identify predictive parameters of poor-performance. Fifty horses underwent thorough clinical and ancillary examinations, including haematological and biochemical evaluation, Doppler echocardiography, standardised exercise tests (SETs) on both treadmill and racetrack, treadmill video-endoscopy and collection of respiratory fluids. Most of the poorly-performing horses exhibited many concomitant diseases. The most frequently diagnosed problems involved the lower and upper respiratory tract and the musculoskeletal system. Poor-performers had lower speeds at a blood lactate (LA) concentration of 4 mmol/L (V(LA4)) and a heart rate (HR) of 200 bpm (V(200)) on treadmill and racetrack, as well as lower values for haematological parameters, plasma angiotensin-converting enzyme and antioxidants, compared to control horses. Problems of the respiratory system were the most frequently diagnosed sub-clinical diseases affecting performance. SETs, together with some blood markers, may be useful as a non-specific diagnostic tool for early detection of diseases that may affect performance.