The effects of Greyhound racing on plasma insulin, glucagon and non-esterified fatty acid have not been extensively studied. We sought to examine the effects of exercise in 19 greyhounds trialed over 400 meters on serum biochemistry, osmolality, nonessential fatty acids, and the major glucoregulatory hormones insulin and glucagon, before and after racing. Due to the non-traditional dietary patterns of many racing Greyhounds, the diet was analyzed for macro and micro-nutrient assessment to determine adequacy of the diet. Many significant biochemical changes were observed and agreed with previous studies, except for low resting glucose, low post-exercise globulin and phosphorus concentrations, and a high CK both before and after exercise. Insulin concentrations increased 10 minutes post-exercise which might be a result of the rise in serum glucose observed. Non-esterified fatty acids decreased 10 minutes post-exercise, as a likely result of the insulin facilitated cellular uptake of fatty acids early in recovery. Glucagon concentrations did not change after exercise despite the rise in insulin. The diet analysis found some mineral deficiencies in sodium, selenium, iodine and copper; and contained a far higher protein and fat content, being atypical of diets that have been shown to improve the performance in racing Greyhounds.