Chromium (Cr) is required for insulin function in the control of cellular glucose uptake. Other functions of Cr relate to its effects on growth, lipid metabolism, immune responses and interactions with nucleic acids. This study was conducted to obtain information on the effect of Cr supplementation on the metabolic responses of five exercising Standardbred horses. During the experiment, horses were fed every day for a 21-day period in a randomized order either a yeast product without Cr (control) or with 4.15 or 8.3 mg Cr day−1. Horses were exercised on a treadmill, alternating a work day of low-speed exercise at 5 m s−1 on a 3% incline for 45 min with a rest day. Each horse was adapted over a 21-day period to his or her respective supplementation before undergoing a standardized exercise test (SET). The SET comprised five incremental steps, each of 4 min duration, on a treadmill with a 3% incline; the first step was at 5 m s−1 and was followed with increments of 1 m s−1. Blood samples were taken for lactate, plasma glucose, serum insulin and cortisol estimation before, during and after each SET (30, 120 min and 24 h post-exercise). Blood Cr was estimated 2 h after feeding the control or Cr-enriched yeast (intake 8.3 mg Cr) in two horses. Heart rate was monitored throughout each SET. Blood lactate and plasma glucose peaks were highest at 8 and 9 m s−1 during the SET when 8.3 mg Cr was supplied. Serum insulin levels declined during the SET and there were no treatment-related changes. Twenty-four hours after exercise, plasma glucose and serum cortisol concentrations returned to basal levels or lower. Serum insulin rebounded 30 min after exercise but 24 h later, serum insulin concentrations were below resting levels. During the recovery period, Cr supplementation did not clearly affect metabolic responses. These results suggest that Cr supplementation had no beneficial effect in healthy, exercising horses.