Background Topical negative pressure (TNP), widely used in wound therapy, is known to stimulate wound edge blood flow, granulation tissue formation, angiogenesis, and revascularization. We have previously shown that application of a TNP of -50 mmHg to the myocardium significantly increases microvascular blood flow in the underlying tissue. We have also shown that a myocardial TNP levels between -75 mmHg and -150 mmHg do not induce microvascular blood flow changes in the underlying myocardium. The present study was designed to elucidate the difference between -25 mmHg and -50 mmHg TNP on microvascular flow in normal and ischemic myocardium. Methods Six pigs underwent median sternotomy. The microvascular blood flow in the myocardium was recorded before and after the application of TNP using laser Doppler flowmetry. Analyses were performed before left anterior descending artery (LAD) occlusion (normal myocardium), and after 20 minutes of LAD occlusion (ischemic myocardium). Results A TNP of -25 mmHg significantly increased microvascular blood flow in both normal (from 263.3 ± 62.8 PU before, to 380.0 ± 80.6 PU after TNP application, * p = 0.03) and ischemic myocardium (from 58.8 ± 17.7 PU before, to 85.8 ± 20.9 PU after TNP application, * p = 0.04). A TNP of -50 mmHg also significantly increased microvascular blood flow in both normal (from 174.2 ± 20.8 PU before, to 240.0 ± 34.4 PU after TNP application, * p = 0.02) and ischemic myocardium (from 44.5 ± 14.0 PU before, to 106.2 ± 26.6 PU after TNP application, ** p = 0.01). Conclusion Topical negative pressure of -25 mmHg and -50 mmHg both induced a significant increase in microvascular blood flow in normal and in ischemic myocardium. The increase in microvascular blood flow was larger when using -25 mmHg on normal myocardium, and was larger when using -50 mmHg on ischemic myocardium; however these differences were not statistically significant.