Use of binocular night vision devices by military aircrew has been associated with visual fatigue. Misalignment between the two binocular images may be one source of this fatigue and could degrade task performance. The eyes have some degree of tolerance to optical misalignment; however, published tolerance limits vary widely and may have limited relevance for military pilots flying long missions. We used a simulated flying task to investigate the effect of misalignment on task performance and visual fatigue. A simulated helicopter flying task was presented simultaneously with three secondary tasks relevant to the visual, auditory, and cognitive demands experience by military aircrew. Task performance was objectively assessed using tracking errors, response times and incorrect responses. Eight participants were exposed to a controlled level of optical misalignment by attaching customised lenses to a Tobii Pro 2 eye tracker. The misaligned condition was compared with an aligned condition with identical workload. Pupil diameter, peripheral skin temperature and ECG data were collected during the task as objective markers of fatigue. Our results showed that misalignment induced significant degradation of task performance both in terms of longer response times and an increased number of incorrect responses. Misalignment was also associated with significantly increased fatigue as measured by reduction of peripheral skin temperature and pupil diameter. Moreover, typical task related modulations in heart rate and heart rate variability were significantly impaired in the misaligned condition.