Abstract Background Disturbances in coronary microcirculatory function, such as the endothelial glycocalyx, are early hallmarks in the development of obesity and insulin resistance. Accordingly, in the present study myocardial microcirculatory perfusion during rest and stress was assessed following metformin or sulodexide therapy in a rat model of diet-induced obesity. Additionally, the effect of degradation of the glycocalyx on myocardial perfusion was assessed in chow-fed rats. Methods Rats were fed a high fat diet (HFD) for 8 weeks and were divided into a group without therapy, and groups that received the anti-diabetic drug metformin or the glycocalyx-stabilizing drug sulodexide in their drinking water during the last 4 weeks of the feeding period. Myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle. In an acute experimental setting, hyaluronidase was administered to chow-fed control rats to determine the effect of enzymatical degradation of the glycocalyx on myocardial perfusion. Results HFD-rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. We confirmed our earlier findings that the robust increase in myocardial perfusion in chow-fed rats after an adenosine challenge (+56%, p = 0.002) is blunted in HFD rats (+8%, p = 0.68). In contrast, 4-weeks treatment with metformin or sulodexide partly restored the increase in myocardial perfusion during adenosine infusion in HFD rats (+81%, p = 0.002 and +37%, p = 0.02, respectively). Treating chow-fed rats acutely with hyaluronidase, to enzymatically degrade the glyocalyx, completely blunted the increase in myocardial perfusion during stress. Conclusions In early stages of HFD-induced insulin resistance myocardial perfusion becomes compromised, a process that can be countered by treatment with both metformin and sulodexide. The adverse effect of acute glycocalyx degradation and protective effect of long-term sulodexide administration on myocardial perfusion provides indirect evidence, suggesting a role for the glycocalyx in preserving coronary microvascular function in pre-diabetic animals.