Background: No-reflow may occur early after reperfusion of ischemic tissues. In a rat model of hepatic ischemia and reperfusion (IR), delayed multifocal no-reflow was observed hours after initial restoration of circulation. We herein sought to characterize this novel phenomenon and investigate its underlying pathophysiological mechanism. Methods: Hepatic IR of male wistar rats was induced by clamping hepatic artery and portal vein for 60 min, followed by release of the clamp for reperfusion. Intravital optical microscopy was employed to image microcirculation (FITC-dextran), cellular hypoxia-reoxygenation (autofluorescence of mitochondrial flavoproteins), extracellular DNA (propidium iodide, PI) and the interactions among leukocytes, platelets and endothelium. Results: Multifocal no-reflow was observed in vivo at the early stage of reperfusion revealed by no recovery of microcirculation and cellular autofluorescence. Interestingly, such no-reflow phenomenon recurred sporadically and remittently about 3 h after initial restoration of circulation. This novel secondary no-reflow was associated with recurrent microcirculatory obstruction caused by stagnation of blood cells. Such obstruction was triggered by activated leukocytes as evidenced by their adhesion with platelets and migration along endothelium. Extracellular DNA near the obstructed sinusoids indicates involvement of neutrophil-extracellular traps (NET). Conclusions: Secondary multifocal no-reflow may occur hours after initial reperfusion. With activated leukocytes and NET playing key roles, the pathogenic mechanism differs distinctly from that of immediate no-reflow and may implicate alternative targets for therapeutic interventions.