In this study, we measured the stiffness of skeletal muscle myofibrils in rigor. Using a custom-built atomic force microscope, myofibrils were first placed in a rigor state then stretched and shortened at different displacements (0.1-0.3 mu m per sarcomere) and nominal speeds (0.4 and 0.8 mu m/s). During stretching, the myofibril stiffness was independent of both displacement and speed (average of 987 nN/mu m). During shortening, the myofibril stiffness was independent of displacement, but dependent on speed (1234 nN/mu m at 0.4 mu m/s; 1106 nN/mu m at 0.8 mu m/s). Furthermore, the myofibril stiffness during shortening was greater than that during stretching and the difference depended on speed (31 % at 0.4 mu m/s; 8% at 0.8 mu m/s). The results suggest that the myofibrils exhibit nonlinear viscoelastic properties that may be derived from myofibril filaments, similar to what has been observed in muscle fibers.