Using the indentation method, we investigated the effects of physical exercise (treadmill running 4 km/day for 15 weeks) on stiffness of the articular cartilage in the canine knee. Considering cartilage to be an elastic material with homogeneity and isotropy, we calculated elastic moduli for femoral, tibial, and patellar cartilages using instant and 15-s deformations after load application. Although the elastic moduli do not represent, because of the non-equilibrium condition, true elastic properties of articular cartilage, they characterize, together with the retardation time spectrum, the integrated response of articular cartilage during the first 15 s after load application. The moderate loading used in our training program caused no macroscopic changes on the articular surface. In running dogs, the mean stiffness of articular cartilage increased by 6% as compared with the controls. Stiffening, which was attributed to the decreased fluid flow in the cartilage, was significant (P less than 0.05) on the patellar surface of the femur and on the tibial condyles. In general, stiffness increased more (approximately 10%) in the cartilage areas, which were repeatedly heavily loaded during running exercise. This alteration in the biomechanical property as well as an increase (approximately 11%) in thickness were considered to be typical responses of articular cartilage to an increased, but physiologic loading pattern.