Chronic atherosclerosis may cause cerebral hypoperfusion and inadequate brain oxygenation, contributing to the progression of cognitive decline. In this study, we exploited two-photon phosphorescence lifetime microscopy to measure the absolute partial pressure of oxygen (PO2) in cortical tissue in both young and old LDLR-/-, hApoB100+/+ mice, spontaneously developing atherosclerosis with age. Capillary red-blood-cell (RBC) speed, flux, hematocrit and capillary diameter were also measured by two-photon imaging of FITC-labelled blood plasma. Our results show positive correlations between RBC speed, flux, diameter and capillary-adjacent tissue PO2. When compared to the young mice, we observed lower tissue PO2, lower RBC speed and flux, and smaller capillary diameter in the old atherosclerotic mice. The old mice also exhibited a higher spatial heterogeneity of tissue PO2, and RBC speed and flux, suggesting a less efficient oxygen extraction.