Dysfunction of the coronary microvasculature has been proposed as one of the important contributors to the diabetes mellitus (DM) associated coronary artery disease (CAD). How different aspects of such dysfunction manifest during disease progression remains incompletely understood. Therefore we studied the time course of coronary microvascular dysfunction in a large animal model of metabolic derangement. Swine with streptozotocin-induced DM type II were fed a high fat diet for up to 15 mo. Coronary small arteries (300 μm) were studied in vitro at 2mo (n=6), 6mo (n=7) and 15 mo (n=5) follow up and compared to healthy controls (n=12, Control). All diabetic groups showed marked hyperglycemia (16±3, 18±4 and 18±1 mmol/l at 2mo, 6mo and 15mo resp.) and hypercholesterolemia (14±2, 17±2 and 18±3 mmol/l) as compared to Control (glucose: 5±1 and cholesterol: 2.0±0.3 mmol/l; all P<0.05). Microvessels showed small plaques only in the 15mo DM group. All DM groups showed increased microvascular stiffness with preserved wall/lumen ratio as compared to control animals.The 2mo and 6mo, but not the 15mo, DM groups showed impaired endothelium-dependent vasodilation to bradykinin, which appeared to result from reduced NO, resp. EDHF bioavailability. Interestingly, the vasoconstriction to endothelin-1 (ET-1) was significantly impaired in the 2mo group (impaired ETA constriction), maintained in the 6mo group, but significantly increased in the 15mo group (reversal ETB dilation to constriction) as compared to Control. The 15mo group also showed increased ETA and ETB expression. Vasodilation to the exogenous NO-donor (SNAP) was similar between groups. In conclusion, specific aspects of CMVD in DM with regard to NO and ET-1 systems are time-dependent. While impaired endothelium-dependent vasodilation due to altered NO/EDHF bioavailability dominates early on, prolonged exposure to high glucose and cholesterol shifts the balance towards increased ET-1 induced vasoconstriction at later stages during CAD development in DM.