The conventional microscope has the disadvantage of a potentially unergonomic posture for the surgeon, which can affect performance. Monitor-based exoscopes could provide a more ergonomic posture, as already shown in pre-clinical studies. The aim of this study was to test the usability and comfort of a novel head-mounted display (HMD)-based exoscope on spinal surgical approaches in a simulated OR setting. A total of 21 neurosurgeons naïve to the device were participated in this prospective trial. After a standardized training session with the device, participants were asked to perform a single-level thoracolumbar decompression surgery on human cadavers using the exoscope. Subsequently, all participants completed a comfort and safety questionnaire. For the objective evaluation of the performance, all interventions were videotaped and analyzed. Twelve men and nine women with a mean age of 34 (range: 24-57) were participating in the study. Average time for decompression was 15 min (IqR 9.6; 24.2); three participants (14%) terminated the procedure prematurely. In these dropouts, a significantly higher incidence of back/neck pain (p = 0.002 for back, p = 0.046 for neck pain) as well as an increased frequency of HMD readjustments (p = 0.045) and decreased depth perception (p = 0.03) were documented. Overall, the surgeons' satisfaction with the exoscope was 84% (IqR 75; 100). Using a standardized, pre-interventional training, it is possible for exoscope-naïve surgeons to perform sufficient spinal decompression using the HMD-based exoscope with a high satisfaction. However, inaccurate HMD setup prior to the start of the procedure may lead to discomfort and unsatisfactory results.