The mechanisms responsible for the inhibition of shortening velocity that occurs during muscle fatigue have not been completely elucidated. Phosphorylation of the myosin regulatory light chain (RLC) occurs during heavy use, however previous reports on its role in affecting velocity have been equivocal. To further understand the process of fatigue we varied the levels of myosin RLC phosphorylation (from 10% to >50%), the concentrations of protons (from pH 7 to 6.2) and phosphate (from 5 mM to 30 mM), all of which change during fatigue. We measured the mechanics of permeable rabbit psoas fibers at a temperature closer to physiological, 30°C, using a temperature jump protocol to briefly activate the fibers at the higher temperature in order to preserve sarcomere homogeneity. While lowered pH alone had an effect on velocity, it was the three factors together, i.e. high phosphorylation, low pH and high phosphate, which acted synergistically to inhibit fiber velocity by approx. 40%. Our data demonstrate that, in conditions that simulate physiological muscle fatigue, myosin phosphorylation does contribute to the inhibition of contraction velocity of fully activated fast muscle fibers.