The feasibility of real-time portal imaging during radiation therapy, through the Cherenkov emission (CE) effect is investigated via a medical linear accelerator (CyberKnife®) irradiating a partially-filled water tank with a 60 mm circular beam. A graticule of lead/plywood and a number of tissue equivalent materials were alternatively placed at the beam entrance face while the induced CE at the exit face was imaged using a gated electron-multiplying-intensified-charged-coupled device (emICCD) for both stationary and dynamic scenarios. This was replicated on an Elekta Synergy® linear accelerator with portal images acquired using the iViewGT™ system. Profiles across the acquired portal images were analysed to reveal the potential resolution and contrast limits of this novel CE based portal imaging technique and compared against the current standard. The CE resolution study revealed that using the lead/plywood graticule, separations down to 3.4 ± 0.5 mm can be resolved. A 28 mm thick tissue-equivalent rod with electron density of 1.69 relative to water demonstrated a CE contrast of 15% through air and 14% through water sections, as compared to a corresponding contrast of 19% and 12% using the iViewGT™ system. For dynamic scenarios, video rate imaging with 30 frames per second was achieved. It is demonstrated that CE-based portal imaging is feasible to identify both stationary and dynamic objects within a CyberKnife® radiotherapy treatment field.