Abstract Objective—To characterize the physiologic response to IV bolus injection of glucose and insulin for development of a combined glucose-insulin test (CGIT) in horses. Animals—6 healthy mares and 1 mare each with pituitary adenoma and urolithiasis. Procedure—Horses were given a CGIT (glucose, 150 mg/kg; insulin, 0.1 U/kg); results were compared with a singular IV glucose tolerance test (GTT; 150 mg/kg) and a singular IV insulin sensitivity test (IST; 0.1 U/kg). Healthy horses were also given a CGIT after receiving xylazine and undergoing stress. Results—Physiologically, the CGIT resulted in a 2-phase curve with positive (hyperglycemic) and negative (hypoglycemic) portions; the positive phase came first (250% of baseline at 1 minute). The descending segment declined linearly to baseline by approximately 30 minutes and to a nadir at 58% of baseline by 75 minutes. After a 35-minute valley, a linear ascent to baseline began. Addition of insulin in the CGIT increased glucose utilization by approximately 4.5 times during the positive phase but not during the negative phase. The diseases' effects and experimental inhibition of insulin secretion with xylazine and stress were detectable by use of the 2 phases of the CGIT. Only a single positive phase resulted from the GTT and a single negative phase from the IST. Conclusions and Clinical Relevance—The CGIT resulted in a consistent, well-defined glycemia profile, which can be disrupted experimentally or by a disease process. The CGIT has clinical potential because it provides integrated information and more information than either the singular GTT or IST. (Am J Vet Res 2005;66:1598–1604)