The oxygen-carrying blood substitute based on polymerized bovine hemoglobin (PBH, Oxyglobin^TM, Biopure Inc., Cambridge, MA) was used to determine efficacy in maintaining tissue pO₂ after an 80% isovolemic blood exchange leading to hematocrit 19.5 ± 0.6% (5.4 ± 0.8 g/dl of hemoglobin (Hb) in red blood cells, and 6.3 ± 0.4 g Hb/dl in plasma). Effects were studied in terms of oxygen delivery, oxygen extraction and tissue pO₂ at the microcirculatory level at 1, 12 and 24 hours after exchange transfusion, while monitoring plasma concentration of PBH in awake hamsters prepared with a window chamber model. At 1 hour after exchange arteriolar (A) and venular (V) diameters were statistically reduced from baseline (1.00), being A 0.78 ± 0.11 and V 0.86 ± 0.10. Arteriolar diameter did not fully recover at 12 hours after exchange (A 0.88 ± 0.10), but venular diameter returned to normal. At 24 hours after exchange arteriolar and venular diameters were not different from baseline. Arteriolar and venular flow velocities were significantly decreased from baseline being A 0.70 ± 0.23 and V 0.76 ± 0.22 at 1 hour post exchange. After 12 and 24 hours, arteriolar and venular flow velocities recovered. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 hour after exchange arteriolar and venular flow was reduced from baseline, being A 0.43 ± 0.28 and V 0.55 ± 0.29. Arteriolar flow was significantly lower at 12 hours after exchange, A 0.79 ± 0.34 of baseline, and recovered after 24 hours. Venular flow was not different from baseline at 12 and 24 hours post exchange. The number of capillaries with red blood cell (RBC) passage (functional capillary density, FCD) at 1 hour after exchange with PBH (0.65 ± 0.7, P < 0.05) was significantly lower than baseline. FCD remained significantly decreased at time point 12 hours (0.84 ± 0.6, P < 0.05). At 24 hours after the exchange transfusion FCD was fully recovered (0.97 ± 0.06 of baseline) and plasma Hb was 2.8 ± 0.3 g/dl. Tissue pO₂ was maximal and significantly above baseline at 1 hour after exchange and decreased progressively at 12 and 24 hours post exchange. Oxygen release to the tissue was minimal at 1 hour and increased at 12 and 24 hours after exchange. These results suggest the impairment of tissue oxygen metabolism following the introduction of PBH in the circulation, which is mitigated as the concentration of PBH declines.