Stress while operating is an important contributor to surgeon health and burnout. Measuring stress is key to improving surgeon and patient outcomes, however biological responses to stress during surgery are variable and difficult to interpret. Participant reported measures of stress have been suggested as an alternative, but the most appropriate measure has not been defined. This study's primary aim was to assess measures of anxiety, stress, and workload before and after surgical simulation and characterize the relationship between these measures. Surgeons completed three laparoscopic exercises from the fundamentals of laparoscopy program (peg transfer, pattern cutting, intracorporeal suturing) in a neutral environment and "stressed" environment (ergonomic, noise, or time pressure). State trait anxiety and self-reported stress on a visual analogue scale were collected prior to simulation and again immediately afterwards. The NASA task load index (TLX) was also administered post-simulation. Of the 26 participants from gynecological and general surgery specialties, state anxiety increased in 98/148 simulations (62%) with a significant mean increase during simulation (32.9 ± 7.9 vs 39.4 ± 10.2, p < .001). Self-reported stress increased in 107/148 simulations (72%), with a significant increase in mean scores during simulation (38.7 ± 22.5 vs 48.9 ± 23.7, p < .001). NASA-TLX scores immediately after simulation ranged from 40 to 118 (mean 60.5 ± 28.7). Greater anxiety and stress scores were reported in "stressed" simulations (43.6 ± 23.1 vs 54.2 ± 23.3; 68.7 ± 27.0 vs 52.4 ± 28.2 respectively) with a significant interaction effect of the "stressed" environment and type of exercise. Anxiety and stress were moderately positively correlated prior to simulation (r = .40) and strongly positively correlated post-simulation (r = .70), however only stress was strongly correlated to workload (r = .79). Stress and anxiety varied by type of laparoscopic exercise and simulation environment. Correlations between anxiety and stress are stronger post-simulation than prior to simulation. Stress, but not anxiety, is highly correlated with workload.