We hypothesized that normotensive sepsis affects the ability of the microcirculation to appropriately regulate microregional red blood cell (RBC) flux. An extensor digitorum longus muscle preparation for intravital study was used to compare the distribution of RBC flux and the functional hyperemic response in SHAM rats and rats made septic by cecal ligation and perforation (CLP). Using intravital microscopy, we found that sepsis was associated with a 36% reduction in perfused capillary density (from 35.3 +/- 1.5 to 22.5 +/- 1.0 capillaries/mm of test line) and a 265% increase in stopped-flow capillaries (from 0.9 +/- 0.2 to 3.3 +/- 0.4 capillaries/mm); the spatial distribution of perfused capillaries was also 72% more heterogeneous. Mean intercapillary distance (ICD) increased 30% (from 25.7 +/- 0.8 to 33.5 +/- 1.6 microns), and the proportion of capillary pairs with intercapillary distances > 33.8 microns (the 75th percentile of ICDSHAM) was greater with sepsis. Mean capillary RBC velocity increased 17% in CLP rats (391 vs 333 microns/s). Laser Doppler flowmetry was used to assess the functional hyperemic response of the extensor digitorum longus muscle before and after a period of maximal twitch contraction designed to increase oxygen demand. RBC flux was 36% lower in the CLP rats at rest. After contraction, RBC flux increased in both SHAM and CLP rats; however, the relative increase was less in the CLP group. We concluded that sepsis affects the ability of the skeletal muscle microcirculation to appropriately distribute RBC flux and to respond to increases in oxygen need.