Background: Promotion of active commuting provides substantial health and environmental benefits by influencing air pollution, physical activity, accidents, and noise. However, studies evaluating intervention and policies on a mode shift from motorized transport to cycling have estimated health impacts with varying validity and precision. Objective: To review and discuss the estimation of air pollution exposure and its impacts in health impact assessment studies of a shift in transport from cars to bicycles in order to guide future assessments. Methods: A systematic database search of PubMed was done primarily for articles published from January 2000 to May 2016 according to PRISMA guidelines. Results: We identified 18 studies of health impact assessment of change in transport mode. Most studies investigated future hypothetical scenarios of increased cycling. The impact on the general population was estimated using a comparative risk assessment approach in the majority of these studies, whereas some used previously published cost estimates. Air pollution exposure during cycling was estimated based on the ventilation rate, the pollutant concentration, and the trip duration. Most studies employed exposure-response functions from studies comparing background levels of fine particles between cities to estimate the health impacts of local traffic emissions. The effect of air pollution associated with increased cycling contributed small health benefits for the general population, and also only slightly increased risks associated with fine particle exposure among those who shifted to cycling. However, studies calculating health impacts based on exposure-response functions for ozone, black carbon or nitrogen oxides found larger effects attributed to changes in air pollution exposure. Conclusion: A large discrepancy between studies was observed due to different health impact assessment approaches, different assumptions for calculation of inhaled dose and different selection of dose-response functions. This kind of assessments would improve from more holistic approaches using more specific exposure- response functions.