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The purpose of this study was to investigate how a given flight task manifested in pilots’ eye behavior and electrodermal activity and how they changed over the course of an instrument flight session. This study implemented a quasi-experimental one-group time series design to identify attentional and emotional states of pilots. Pilots were emotionally aroused during flight segments like takeoff and climb, approach and landing-1, missed approach, and approach and landing-2. Pilots felt visually demanding during takeoff and climb, approach and landing-1, missed approach, en route-2, and approach and landing-2. Missed approach was the most visually taxing flight segment, where pilots rapidly shifted their attention from one fixation to another on their primary flight display (PFD). The research findings from this human-in-the-loop experiment inform the aviation community about the precise evaluation of pilots’ mental workload during flight.