Subchondral bone injuries often precede articular cartilage damage in osteoarthritis and are common in thoroughbred racehorses due to the accumulation of fatigue damage from high speed racing and training. Thus, racehorses provide a model to investigate the role of subchondral bone in joint disease. We assessed the association of horse and racing related factors and micro-CT based micromorphology of three separate subchondral bone layers with the initial stiffness and compressive fatigue life of bone plugs. Furthermore, we investigated three different definitions of fatigue failure of subchondral bone during compressive fatigue testing. Initial stiffness was 2,362 ± 443 MPa (mean ± standard deviation). Median compressive fatigue life during cyclic loading to -78 MPa was 16,879 (range 210 to 57,064). Subchondral bone stiffness increased over a median of 24% (range 3%–42%) of fatigue life to a maximum of 3,614 ± 635 MPa. Compressive fatigue life was positively associated with bone volume fraction in the deeper layers of subchondral bone, maximal stiffness, and the number of cycles to maximal stiffness. Initial stiffness was positively associated with tissue mineral density in the deeper layers and bone volume fraction in the superficial layer. Most specimens with a fatigue life of less than 5,500 cycles fractured grossly before reaching 30% reduction of maximal stiffness. Cycles to 10% reduction of maximal stiffness correlated strongly with cycles to lowest recorded stiffness at gross fracture and thus is a valid alternative failure definition for compressive fatigue testing of subchondral bone. Our results show that subchondral bone sclerosis as a result of high speed exercise and measured as bone volume fraction is positively associated with compressive fatigue life and thus has a protective effect on subchondral bone. Further research is required to reconcile this finding with the common collocation of fatigue damage in sclerotic subchondral bone of racehorses.