Muscle, motor unit and muscle fibre type-specific differences in force-generating capacity have been investigated for many years, but there is still no consensus regarding specific differences between slow- and fast-twitch muscles, motor units or muscle fibres. This is probably related to a number of different confounding factors disguising the function of the molecular motor protein myosin. We have therefore studied the force-generating capacity of specific myosin isoforms or combination of isoforms extracted from short single human muscle fibre segments in a modified single fibre myosin in vitro motility assay, in which an internal load (actin-binding protein) was added in different concentrations to evaluate the force-generating capacity. The force indices were the x-axis intercept and the slope of the relationship between the fraction of moving filaments and the α-actinin concentration. The force-generating capacity of the β/slow myosin isoform (type I) was weaker (P < 0.05) than the fast myosin isoform (type II), but the force-generating capacity of the different human fast myosin isoforms types IIa and IIx or a combination of both (IIax) were indistinguishable. A single fibre in vitro motility assay for both speed and force of specific myosin isoforms is described and used to measure the difference in force-generating capacity between fast and slow human myosin isoforms. The assay is proposed as a useful tool for clinical studies on the effects on muscle function of specific mutations or post-translational modifications of myosin.Myosin is the molecular motor protein in skeletal muscle that generates force and movement. It is expressed in multiple isoforms that have different enzymatic properties. There are isoform specific differences in contractile speed, but there is no consensus if the force generating capacity differs between isoforms. In this study we have modified a single fibre in vitro motility assay to measure both force and speed generated by specific myosin isoforms extracted from short single human muscle fibre segments. It is shown that human slow myosin is weaker and slower than fast myosin. This assay is put forward as a useful tool for future investigations on myosin function in response to modifications associated with muscle disease or ageing.