Despite preclinical evidence that nitric oxide (NO) donors influence both tumor perfusion and hypoxia, in clinical trials nitroglycerin has not been shown to improve the treatment results of all patients with non-small cell lung cancer (NSCLC). Biomarkers are therefore needed to select patients for treatment with NO donors. In this window-of-opportunity study we demonstrate the effect of nitroglycerin on hypoxia in patients using repeated hypoxia PET-imaging: we observed a reduction of hypoxia - quantified by uptake of PET tracer HX4- of varying magnitude upon application of a nitroglycerin..