Passenger and flight crew concerns over the negative impact of the aircraft cabin environment on health and comfort have lingered for years. During flight, passengers and crew can experience reduced atmospheric pressure, low relative humidity, and potential air quality degradation, which could lead to adverse health complaints. The goal of this project was to investigate the potential link between perceived health symptoms and discomfort and aircraft cabin environmental conditions and human factors. Passengers and crew completed validated health and comfort surveys on 130 domestic and international flights across varied aircraft, durations, and seasons. Environmental conditions and airborne levels of contaminants were measured during 80 of these flights. Extremes in cabin temperature or degraded air quality parameters led to higher rates of passenger discomfort. Odds ratios were higher for several adverse health symptoms for certain aircraft types and durations, while ventilation rates were positively correlated with discomfort and adverse health symptoms. Median levels of CO, CO2, and ozone were below established exposure limits. Results from this study help further our knowledge of aircraft comfort and can help airlines and industry agencies determine the most critical impacts on health and comfort to better inform standards to improve passenger and crew experiences.