Neuromuscular function is impaired following an unaccustomed bout of eccentric exercise. However, through the repeated bout effect (RBE), the muscle is protected from impaired neuromuscular function following a subsequent bout of eccentric exercise. It has been speculated that the addition of sarcomeres in series (sarcomerogenesis) contributes to the RBE by reducing mechanical strain on muscle fibers during active lengthening. However, whether sarcomerogenesis actually contributes to the RBE is unknown. We investigated whether a single bout of damaging eccentric exercise induces serial sarcomerogenesis, and if this offers a protective effect on the muscle. Using an in-vivo set up, twenty-four Sprague-Dawley rats performed maximal eccentric contractions of the plantar flexors to impair mechanical function. Thirteen days following the initial eccentric exercise bout, twelve rats were sacrificed, to assess serial sarcomere number (SSN) of the soleus and medial gastrocnemius (MG) via laser diffraction. The remaining twelve rats completed an identical second bout of eccentric exercise to assess the RBE. A single bout caused long lasting impairments in torque production (−3% for 100 Hz;​ −16 % for 10 Hz;​ P < 0.05 compared to baseline). Following the repeated bout, there was a protective effect with all torque measures recovering by 2 days post-exercise (P > 0.05 compared to baseline). SSN did not differ between the control and exercised legs for either muscle (P > 0.05). There was a robust RBE following the second bout of eccentric exercise, with no increase in SSN indicating serial sarcomerogenesis is not one of the primary initial mechanisms contributing to the RBE.