We examined the adaptation of plantar flexor muscles of female rats to 6 weeks (5 days/week) of lengthening contractions. After repeated lengthening contractions, a decrease in myofiber area of gastrocnemius medialis (26%) was accompanied by an increase in extracellular matrix (ECM) (42%) and collagen content (30.9%) without changes in muscle mass. Decrease in myofiber area (13%) and muscle mass of soleus (19%) was associated with increased collagen content (28%) and ECM (15%). Relative number of soleus myofibers stained for fast myosin increased by 26%. For plantaris, increases in collagen content (32.3%), percent ECM (17%), and myofiber area (6%) were recorded. We also observed (1) increases (3.3%) in the collagen content of the Achilles tendon, (2) no change in the crosslink content of any of the tissues tested, and (3) no difference in the force-frequency relationship of the plantar flexor muscles. Substantial decreases in myofiber areas with increases in muscle connective tissue by 6 weeks of repeated lengthening contractions did not appear to result in isometric force loss.