Introduction: The suitability of EndoSequence BC Sealer (BC Sealer; Brasseler USA, Savannah, GA) for warm vertical compaction has been questioned. The aim was to evaluate the cytotoxicity and the effect of heating on the physicochemical properties of a new calciumbased root canal sealer (EndoSequence BC Sealer HiFlow [HiFlow]) in comparison with EndoSequence BC Sealer. Methods: Human periodontal ligament fibroblasts were incubated for 1, 2, or 3 days with material extracts of different concentrations, and cell viability was evaluated by Cell Counting Kit-8 (Enzo Life Sciences Inc, Burlington, Ontario, Canada). The setting time, flow, film thickness, microhardness, radiopacity, and radiopacity of the 2 sealers were measured according to ISO 6786/2012. The continuous changes in viscosity were tested by a stress-controlled rheometer at shear rates ranging from 0.01-10 s(-1) and different temperatures, and chemical composition was assessed by Fourier-transform infrared spectroscopy. Results: Cell viability was significantly decreased on day 3 for the 1:4 diluted extract from both materials. The setting time, microhardness, and solubility of HiFlow were similar to BC Sealer at 37 degrees C and 100 degrees C. HiFlow had significantly higher flow and radiopacity than BC Sealer at room temperature (P < .05), and when heated, HiFlow retained its higher flow and lower film thickness (P < .05). Both sealers showed decreasing viscosity with increasing shear rate, and at a shear rate of 0.01 and 0.1 s(-1), HiFlow exhibited lower viscosity than BC Sealer at all temperatures measured. The chemical composition of the 2 sealers was not changed by heating. Conclusions: HiFlow showed better performance on flow/viscosity and film thickness, especially under high temperatures, which are generated by the commonly used warm vertical compaction technique.