The aim of this study was to document the physical stress experienced by a surgeon during thoracoscopic pulmonary lobectomy and mediastinal lymph node dissection for lung cancer by measuring the intraoperative electromyography (EMG). Surface EMG was recorded during 12 cases of thoracoscopic lobectomy. During the operation, 16 channels of a wireless EMG were used to measure muscle activity and fatigue from the bilateral muscles of the splenius capitis (SC), upper trapezius (UT), middle deltoid (MD), flexor carpi radialis (FCR), extensor carpi radialis (ECR), lumbar erector spinae (LES), rectus femoralis (RF), and tibialis anterior (TA). The EMG signals were processed to collect the values of the root mean square for muscle activity and median frequency (MF) for muscle fatigue. All operations were completed without adverse events. The mean operating time was 99.16±35.15 minutes. During the operation, the mean muscle activity of all muscles was 21.91±12.85 mV. High muscle activity was observed in the bilateral FCR and ECR, whereas low muscle activity was observed in the bilateral SC and LES. The final MFs in the bilateral SC and LES were found to be decreased from the initial status, which implied increased muscle fatigue. The muscles of the right and left LES were significantly fatigued by up to 29% and 37% compared to their initial status (P=0.021 and P=0.007, respectively). The MFs of the bilateral LES decreased with time (an average decreases of 0.008/5 minutes, P=0.002 in right LES and 0.004/5 minutes, P=0.018 in left LES). During thoracoscopic lobectomy, muscle fatigue was observed in muscles related to a static posture, such as the bilateral SC, UT, and ES. Further studies are required to investigate the ergonomic adjustments needed to reduce muscle fatigue in these static muscles.