The purpose of this study was to determine the effects of a novel combination of mechanical activity paradigms on the isomyosin distribution in rat hindlimb muscles. Thirty female Sprague-Dawley rats were divided into five experimental groups as follows: normal control, functional overload (OV) of the plantaris, OV in conjunction with hindlimb suspension (OV-S), and a combination of OV-S and either static standing weight-bearing activity (OV-SS) or high-incline treadmill exercise (OV-SE). OV of the plantaris resulted in significant hypertrophy and significant fast-to-slow isomyosin shifts. These changes were completely inhibited by the addition of hindlimb suspension (OV-S). Also, neither of the two weight-bearing regimes (OV-SS and OV-SE) was able to attenuate the suspension-induced atrophy. In the vastus intermedius and vastus lateralis, however, OV-SS was able to partially retard the atrophy associated with suspension. In both the plantaris and vastus intermedius, only OV-SS was able to partially reverse the slow-to-fast isomyosin transitions associated with suspension. These results suggest that the type of mechanical activity is important in determining adaptation to altered loading conditions, with OV-SS appearing more effective than OV-SE in reversing the effects of unweighting.