Muscle performance is improved after a brief period of exercise (warm-up). One factor that is known to strongly affect force production is the myoplasmic concentration of inorganic phosphate ([P(i)]). Improved performance after warm-up may therefore be due to a reduction of [P(i)]. Herein, we show that after a warm-up protocol (15 tetani at 2-s intervals), tetanic force is increased by approximately 6% (P < 0.05) and [P(i)] is almost halved (P < 0.05) in isolated mouse soleus muscle. A warm-up protocol with longer intervals (15 tetani at 5-s intervals) reduced tetanic force and did not alter [P(i)]. We conclude that a reduction of [P(i)] contributes to the force-potentiating effect of warm-up.