Direct physicochemical interactions between the major components of electronic cigarette liquids (e-liquids): glycerol (VG) and propylene glycol (PG), and lung surfactant (LS) were studied by determining the dynamic surface tension under a simulated breathing cycle using drop shape method. The studies were performed for a wide range of concentrations based on estimated doses of e-liquid aerosols (up to 2500 × the expected nominal concentrations) and for various VG/PG ratios. The results are discussed as relationships among mean surface tension, surface tension amplitude, and surface rheological properties (dilatational elasticity and viscosity) versus concentration and composition of e-liquid. The results showed that high local concentrations (>200 × higher than the estimated average dose after a single puffing session) may induce measurable changes in biophysical activity of LS; however, only ultra-high e-liquid concentrations inactivated the surfactant. Physiochemical characterization of e-liquids provide additional insights for the safety assessment of electronic nicotine delivery systems (ENDS).