Surgical time-outs are designed to promote situation awareness, teamwork, and error prevention. The pre-incision time-out in particular aims to facilitate shared mental models prior to incision. Objective, unbiased measures to confirm its effectiveness are lacking. We hypothesized that providers’ mental workload would reveal team psychophysiological mirroring during a formal, well-executed pre-incision time-out. Heart rate variability was collected during cardiac surgery cases from the surgeon, anesthesiologist, and perfusionist. Data were analyzed for six cases from patient arrival until sternal closure. Annotation of surgical phases was completed according to previously developed standardized process models of aortic valve replacement and coronary artery bypass graft procedures, producing thirteen total surgical phases. Statistical analysis revealed significant main effects. Tukey HSD post hoc tests revealed significant differences across provider roles within various phases, including Anesthesia Induction, Heparinization, Initiation of Bypass, Aortic Clamp and Cardioplegia, Anastomoses or Aortotomy, Separation from Bypass, and Sternal Closure. Despite these observed differences between providers over various surgical phases, the Pre-incision Time-out phase revealed almost negligible differences across roles. This preliminary work supports the utility of the pre-incision safety checklist to focus the attention of surgical team members and promote shared team mental models, measured via psychophysiological mirroring, using an objective mental workload measure. Future studies should investigate the relationship between psychophysiological mirroring among surgical team members and the effectiveness of the pre-incision time-out checklist.