In this study, we simultaneously measured nitric oxide (NO) and oxygen (O2) dynamics in the myocardium during myocardial ischemia–reperfusion (IR) utilizing sol–gel modified electrochemical NO and O2 microsensors. In addition, we attempted to clarify the correlation between NO release in the ischemic period and O2 restoration in the myocardium after reperfusion, comparing a control heart with a remote ischemic preconditioning (RIPC)-treated heart as an attractive strategy for myocardial protection. Rat hearts were randomly divided into two groups: a control group (n=5) and an RIPC group (n=5, with RIPC treatment). Myocardia that underwent RIPC treatment (182±70nM, p<0.05) released more NO during the ischemic period than those of the control group (63±41nM). The restoration value of oxygen tension (pO2) in the RIPC group significantly increased and was restored to pre-ischemic levels (92.6±36.8%); however, the pO2 of the control group did not increase throughout the reperfusion period (5.7±7.5%, p=0.001). Myocardial infarct size measurements revealed a significant decrease in cell death in the myocardium region of the RIPC group (41.44±6.42%, p=0.001) compared with the control group (60.05±10.91%). As a result, we showed that the cardioprotective effect of RIPC could be attributed to endogenous NO production during the ischemic period, which subsequently promoted reoxygenation in post-ischemic myocardia during early reperfusion. Our results suggest that the promotion of endogenous formation during an ischemic episode might be helpful as a therapeutic strategy for protecting the myocardium from IR injury. Additionally, our NO and O2 perm-selective microsensors could be utilized to evaluate the effect of drug or treatment.