Hereditary muscular disease is well described in racehorses, yet little is known about traumatic muscle disease associated with unaccustomed exercise or training. The objective of the study was to compare sedentary horses, racehorses undergoing training for the first time (unaccustomed exercise), and experienced racehorses during a training season (accustomed exercise) to investigate the effect of exercise and training on serum muscle enzyme activities and other variables. Horses were sampled prior to exercise for serum activities of aspartate amino transferase (AST), creatine kinase and other variables γ glutamyl transferase (GGT) and serum amyloid A (SAA) in a three-part study. Serum activities of AST and GGT were higher in fit racehorses (n=47) compared with sedentary horses (n=57) at a single time point (P<0.05). The monthly serum activity of AST in two-year-old racehorses (n=10) increased from month 1 to 4 of unaccustomed training (P<0.05). The serum activities of AST and GGT in three-year-old racehorses (n=12) previously accustomed to exercise sampled fortnightly to monthly showed a linear increase (P<0.05) with cumulative training days over the seven months of training, but showed minimal increase during the first four months of accustomed training. SAA was weakly correlated to cumulative training days. In conclusion, AST activity was increased by unaccustomed exercise and cumulative training stress in the racehorse. GGT appeared to be correlated to cumulative training load. Mild to moderate elevations in serum AST in racehorses may be associated with cumulative muscle damage from training or trauma associated with unaccustomed exercise.