Thirteen Standardbred horses trained intensively for 34 weeks and detrained for 12 weeks to investigate the effects of training, overtraining and detraining on muscle metabolites, buffering capacity and enzyme activities (CS, HAD and LDH). After a standardised exercise test to fatigue at 10 m/s (approximately 100% VO2max), there was significant depletion of [ATP], [PCr] and muscle [glycogen] and accumulation of muscle and plasma [lactate], [NH3] and elevated muscle temperature. After training, associated with increased run time to fatigue (148%), there was reduced depletion of muscle [glycogen] and increased [NH3] and muscle temperature at fatigue. Training resulted in increased muscle buffering capacity (19%) and activities of CS (29%) and HAD (32%) and reduced glycogen utilisation (1.32 mmol/s in week 1 to 0.58 mmol/s in week 32). Plasma [lactate] at fatigue increased with training as opposed to muscle [lactate] implying enhanced ability to remove lactate from muscle. Overtraining resulted in reduced run time and associated effects in overtrained horses. While muscle [glycogen] prior to exercise was lower in overtrained horses, glycogen utilisation/s was not reduced and it may not, therefore, have caused the reduced run time. Prolonged high intensity training caused primarily aerobic adaptations and poor performance associated with overtraining may not be due to metabolic disturbances.