Effects of decreasing stimulation frequency on length-force characteristics were determined for rat medial gastrocnemius muscle. The peripheral nerve was stimulated supramaximally with a succession of twitch and frequencies of 100, 50, 40, 30, and 15 Hz. Active peak tetanic and twitch forces and active muscle geometry were analyzed. Optimal muscle length and active slack length shifted significantly (P < 0.05) to higher muscle length by a maximum of 2.8 and 3.2 mm, respectively. Further significant effects were found for distal fiber length and mean sarcomere length of distal fiber (increases) and for fiber angle and aponeurosis length (decreases). Neither muscle length range between active slack and optimal length nor aponeurosis angle was altered significantly. We concluded that decreasing stimulation frequency-dependent length-force characteristics are affected by a complex interaction of length-dependent calcium sensitivity, potentiation of the contractile system, distribution of sarcomere length, and interactions between force exerted and aponeurosis length. Length-dependent calcium sensitivity seems to be a major factor determining the magnitude of the shift of optimal muscle length.