Abstract: The cause of muscle fatigue has been studied for more than 100 years, yet its molecular basis remains poorly understood. Prevailing theories suggest that much of the fatigue-induced loss in force and velocity can be attributed to the inhibitory action of metabolites, principally phosphate (Pi) and hydrogen ions (H, i.e. acidosis), on the contractile proteins, but the precise detail of how this inhibition occurs has been difficult visualize at the molecular level. However, recent technological developments in the areas of biophysics, molecular biology and structural biology are enabling researchers to directly observe the function and dysfunction of muscle contractile proteins at the level of a single molecule. In fact, the first direct evidence that high levels of H and Pi inhibit the function of muscle's molecular motor, myosin, have recently been observed in a single molecule laser trap assay. Likewise advances in structural biology are taking our understanding further, providing detail the atomic level of how some metabolites might alter the internal motions of myosin and thereby inhibit its ability to generate force and motion. Finally, new insights are also being gained into the indirect role that muscle regulatory proteins troponin (Tn) and tropomyosin (Tn) play in the fatigue process. In vitro studies, incorporating TnTm, suggest that a significant portion of the decrease force and motion during fatigue may be mediated through a disruption of the molecular motions of specific regions within Tn and Tm. These recent advances are providing unprecedented molecular insight into the structure and function of the contractile proteins and in the process are reshaping our understanding of the process of fatigue.