IMPORTANCE The use of electronic health records (EHRs) is directly associated with physician burnout. An underlying factor associated with burnout may be EHR-related fatigue owing to insufficient user-centered interface design and suboptimal usability. OBJECTIVE To examine the association between EHR use and fatigue, as measured by pupillometry, and efficiency, as measured by mouse clicks, time, and number of EHR screens, among intensive care unit (ICU) physicians completing a simulation activity in a prominent EHR. DESIGN, SETTING, AND PARTICIPANTS A cross-sectional, simulation-based EHR usability assessment of a leading EHR system was conducted from March 20 to April 5, 2018, among 25 ICU physicians and physician trainees at a southeastern US academic medical center. Participants completed 4 simulation patient cases in the EHR that involved information retrieval and task execution while wearing eye-tracking glasses. Fatigue was quantified through continuous eye pupil data; EHR efficiency was characterized through task completion time, mouse clicks, and EHR screen visits. Data were analyzed from June 1, 2018, to August 31, 2019. MAIN OUTCOMES AND MEASURES Primary outcomes were physician fatigue, measured by pupillometry (with lower scores indicating greater fatigue), and EHR efficiency, measured by task completion times, number of mouse clicks, and number of screens visited during EHR simulation. RESULTS The 25 ICU physicians (13 women; mean [SD] age, 33.2 [6.1] years) who completed a simulation exercise involving 4 patient cases (mean [SD] completion time, 34:43 [11:41] minutes) recorded a total of 14 hours and 27 minutes of EHR activity. All physician participants experienced physiological fatigue at least once during the exercise, and 20 of 25 participants (80%) experienced physiological fatigue within the first 22 minutes of EHR use.