Although muscle strain injury is one of most important issues in sports medicine, there is little known about the molecular events on regeneration process and embryonic formation in vivo injured muscle. The purpose of this study was to examine the effects of lengthening contractions (LCs) on tendon-related markers signaling pathways. We hypothesized that tendon-related markers are important role for the recovery from muscle injury. We employed our originally developed device with two LC modes to modulate the intensity in rat gastrocnemius muscle (1, 2). Since we reported one bout of LCs causes decreases in muscle torque and increase protein degradation signaling pathway and myostatin (3), we hypothesized that LCs cause an increase in fibrosis concomitant with activations of tendon-related markers. Male Wistar rats (n = 18) were randomly divided into fast velocity LCs group (FAST, 180deg/s, n = 6), slow LCs group (SLOW, 30deg/s, n = 6), and control group (control, n = 6). The FAST and SLOW rats were anesthetized with isoflurane (gas flow rate, 450ml/min, concentration, 2.0%). The triceps surae muscle of the right hindlimb was then electrically stimulated with forced isokinetic dorsi-flexion (30°/s and from 0 to 45°). Tissue contents and localizations of tenomodulin, scleraxis, and myostatin were measured by western blotting and immuohistochemistry. The mRNA expression of type I collagen alpha 2 (col1a2) and mohawk was evaluated using real time reverse transcriptasepolymerase chain reaction. One-way ANOVA was used to compare the body mass, muscle mass, protein and mRNA analysis. No significant changes were observed in both body mass and hindlimb muscles between three groups. The torque was significantly lower in FAST than in SLOW (day2; 59.9±17.2 vs. 101.5±22.9 mNm, P < 0.01). Tenomodulin and myostatin and col1a2 mRNA showed significantly enhanced expression in FAST than in the other two groups (tenomodulin; ∼2.5 fold, P < 0.01, myostatin; ∼3.8 fold, P < 0.01, col1a2; ∼8.5 fold, P < 0.05). Immunohistochemical staining in FAST, but not in SLOW, was mainly localized in connective tissues between muscle fibers. On the other hands, scleraxis and mohawk mRNA in SLOW was significantly higher than that in control (scleraxis; ∼2.7 fold, P < 0.01, mohawk; ∼6.7 fold, P < 0.05). We conclude that fast LCs cause an increase in connective tissue fibrosis through the activated myostatin signaling pathway. In addition, the present results suggest that the severity of LCs-induced damage cause different expressions of tendon-related markers.