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BACKGROUND: Aircraft passengers’ physical activity levels are often limited during flight for extended periods of time, which can have serious impact on health, comfort, and passenger experience. However, several factors, such as limited personal space and social factors, can make movement difficult. OBJECTIVE: The study aimed to identify participant behaviour and postures during medium to long-haul flights and their effect on location and severity of physical discomfort. This work investigated how aircraft passengers’ comfort ratings changed over time and explored the association between postures and comfort ratings during flight. The study also explored the association between performing in-flight exercises and factors limiting participants’ abilities to complete these exercises. METHOD: Twenty-nine participants sat in an aircraft simulated cabin for 180 minutes and periodically performed in-seat exercises. They also completed a questionnaire providing data on demographic information, self-reported discomfort scores for multiple areas of the body, which types of exercises participants performed, and qualitative comments about discomfort. Self-reported comfort ratings were measured at 20-min intervals and posture identification was conducted by analysing the video recordings. RESULTS: Six postures were identified, and it was observed that individual participants changed posture at least once and up to nine times during the three-hour period. Body part areas with the highest reported scores of discomfort ratings were: back of the neck, back-left shoulder, back-right shoulder, back-left buttock, and lower back. Participants reported that they had difficulty in performing recommended in-flight exercises due to limited space. CONCLUSIONS: Limited space in aircraft seating restricts passenger movement. Some body areas are more prone to discomfort.