The purpose of the study was to investigate microcirculation and vascular reactivity during experimental endotoxemia and endotoxin tolerance in humans by comparing different methods of approach. Endotoxin tolerance was induced in nine healthy volunteers by intravenous injection of 2 ng · kg−1 · d−1 LPS for 5 consecutive days. Microcirculation and vascular reactivity were monitored before and after LPS administrations on days 1 and 5 by near-infrared spectroscopy, sidestream dark-field imaging, and forearm blood flow by venous occlusion strain-gauge plethysmography during local intra-arterial infusion of endothelial-dependent vasodilator acetylcholine (0.5, 2, and 8 μg · min−1 · dL−1). LPS administration induced a significant rise in all measured cytokines. During subsequent LPS administrations, the increase in cytokine levels was almost completely abolished, indicating the development of tolerance. Near-infrared spectroscopy showed 79% (interquartile range [IQR], 62%-92%) attenuation of recovery slope after ischemia 2 h after LPS administration on day 1 (P = 0.04), which was absent on day 5 (P = 0.72). Sidestream dark-field imaging showed 33% (IQR, 14%-40%) and 30% (IQR, 10%-33%) diminished flow in medium and large microvessels, respectively, 2 h after LPS administration on day 1 (P = 0.07 and 0.04, respectively), which was absent on day 5 (P = 0.47 for both vessels). Forearm blood flow measurements showed an attenuation of acetylcholine-induced vasodilatory response, with 67% (IQR, 45%-72%) 4 h after the first LPS administration (P = 0.01), but not when tolerance was present on day 5 (P = 0.61). Human endotoxemia results in endothelial dysfunction that can be adequately detected with different methods and was restored with development of LPS tolerance.