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Sepsis-induced microvascular leukocyte/endothelial cell interaction may result in a deterioration of capillary perfusion that finally leads to septic organ dysfunction. The aim of the present study was to characterize a novel, sublethal, two-hit model of chronic systemic sepsis that allows the repeated analysis of microcirculation by intravital microscopy. In Syrian golden hamsters the effect of a single i.v. endotoxin (LPS, 2 mg/kg, E. coli) injection (SH-LPS group, n = 5 animals) vs. a double LPS injection (DH-LPS group, n = 6 animals) was analyzed. After monitoring baseline parameters (t1), measurements were performed at 30 min (t2), 3 h (t3), 8 h (t4), 24 h (t5), 48 h (t6), 56 h (t7) and 72 h (t8) (both groups) after initial LPS exposure. In DH-LPS animals, a second LPS injection (2 mg/kg) was given at t6 (48 h). Intravital fluorescence microscopy was performed in a dorsal skin fold chamber preparation and allowed determination of leukocyte-endothelial cell interaction (leukocyte rolling and sticking), and measurement of functional capillary density (FCD), which served as a measure of capillary perfusion. The first LPS injection comparably altered leukocyte/endothelial cell interaction and capillary perfusion in both groups (t1-t6, P > 0.05, MANOVA). Between t6 and t8 leukocyte adherence decreased in SH-LPS animals, whereas in DH-LPS animals adherence remained constantly elevated (SH-LPS: −53.0 ± 6.2% between t6 and t8 vs. DH-LPS: −3 ± 5; P < 0.05). The ongoing inflammatory response in DH-LPS animals was associated with a progressive deterioration of FCD, whereas FCD remained constant in SH-LPS animals (DH-LPS: −71.5 ± 17% between t6 and t8 vs. SH-LPS: 3.0 ± 13%; P < 0.05). In parallel, coagulatory parameters were found significantly altered only in DH-LPS animals but not in SH-LPS animals. We conclude that “double hit” LPS exposure is an appropriate model (i) to analyze repeatedly over time microcirculatory disorders under conditions of persistent endotoxemia-induced inflammatory response, and (ii) to prove the effectiveness of novel anti-inflammatory strategies.