The fatigue life of cortical bone can vary several orders of magnitude, even in identical loading conditions. A portion of this variability is likely related to intracortical microarchitecture and the role of vascular canals as stress concentrators. The size, spatial distribution, and density of canals determine the peak magnitude and volume of stress concentrations. This study utilized a combination of experimental fatigue testing and image-based finite element (FE) analysis to establish the relationship between the stressed volume (i.e., volume of bone above yield stress) associated with vascular canals and the fatigue life of cortical bone. Thirty-six cortical bone samples were prepared from human femora and tibiae from five donors. Samples were allocated to four loading groups, corresponding to stress ranges of 60, 70, 80, and 90 MPa, then cyclically loaded in zero-compression until fracture. Porosity, canal diameter, canal separation, and canal number for each sample was quantified using X-ray microscopy (XRM) after testing. FE models were created from XRM images and used to calculate the stressed volume. Stressed volume was a good predictor of fatigue life, accounting for 67% of the scatter in fatigue-life measurements. An increase in stressed volume was most strongly associated with higher levels of intracortical porosity and larger canal diameters. The findings from this study suggest that a large portion of the fatigue-life variance of cortical bone in zero-compression is driven by intracortical microarchitecture, and that fatigue failure may be predicted by quantifying the stress concentrations associated with vascular canals.