Mucociliary clearance is an important defense mechanism that mediates removal of foreign particles and chemicals from the airways. Cilia beating thereby plays a key role that determines the rate of mucus clearance and thus constitutes a vital function of respiratory epithelia. Cigarette smoke has been reported to adversely impact cilia function in vitro and in vivo, by changing cilia beating frequency (CBF) or impairing ciliogenesis. To monitor CBF, semi-automated methods such as CiliaFA combine high-speed video recording with the ability to determine CBF. Using CiliaFA, we were able to confirm that the MucilAirTM CBF can be modulated in vitro by either 100 μM isoproterenol or a temperature shift to 4°C. Moreover, in vitro exposure of MucilAirTM with whole smoke from conventional cigarettes (3R4F at 0 mg/L, 0.15 mg/L and 0.25 mg/L nicotine) caused a decrease in the total surface area of the culture showing active cilia beating. Cilia on the epithelial cell surface that were detected to be still active after 3R4F exposure, showed variable beat frequencies, ranging from normal to decreased CBF. Compared to 3R4F cigarette smoke exposure, the effect of equivalent concentrations (based on nicotine) of a candidate modified risk tobacco product (MRTP: THS2.2) aerosol was much less pronounced, i.e. the total surface area of cilia beating as well as beating frequency was less impacted by THS2.2. Overall, this study clearly discriminated the effects of THS2.2 from the deleterious impact of 3R4F on cilia beating.