Junctophilins (JP1 and JP2) are expressed in skeletal muscle and are the primary proteins involved in transverse (T)-tubule and sarcoplasmic reticulum (SR) membrane apposition. During the performance of eccentric contractions, the apposition of T-tubule and SR membranes may be disrupted, resulting in excitation-contraction (EC) coupling failure and thus reduced force-producing capacity. In this study, we made three primary observations: 1) through the first 3 days after the performance of 50 eccentric contractions in vivo by the left hindlimb anterior crural muscles of female mice, both JP1 and JP2 were significantly reduced by approximately 50% and 35%, respectively, while no reductions were observed after the performance of nonfatiguing concentric contractions; 2) following the performance of a repeated bout of 50 eccentric contractions in vivo, only JP1 was immediately reduced ( approximately 30%) but recovered by 3-day postinjury in tandem with the recovery of strength and EC coupling; and 3) following the performance of 10 eccentric contractions at either 15 degrees or 35 degrees C by isolated mouse extensor digitorum longus (EDL) muscle, isometric force, EC coupling, and JP1 and JP2 were only reduced after the eccentric contractions performed at 35 degrees C. Regression analysis of JP1 and JP2 content in tibialis anterior and EDL muscles from each set of experiments indicated that JP damage is significantly associated with early (0-3 days) strength deficits after performance of eccentric contractions (R = 0.49; P < 0.001). As a whole, the results of this study indicate that JP damage plays a role in early force deficits due to EC coupling failure following the performance of eccentric contractions.