When diesel soot particles experience high-temperature treatment during exhaust purification aiming to reduce emission, their physicochemical properties and toxicity would change as well. However, rare studies investigated the relationship between properties and health effects of soot under high temperatures. This study compared the physicochemical properties, oxidative potential (OP, dithiothreitol (DTT) method), and cytotoxicity of model diesel soot before and after 300°C and 500°C treatments in air. It was observed that the oxygen-containing functional groups, environmental persistent free radicals, surface area and pore volume increased a lot after the 500°C process, accompanied with the appearance of hollow core and decreased particle size in the images collected by high-resolution transmission electron microscopy, indicating the deep oxidation into carbonaceous core of soot. In addition, the enhancements of OPDTT and cytotoxicity were in accordance with the changes in physicochemical properties. The toxic increment at 500°C was the most due to core oxidation, whether it was compared with 300°C heating or the surface oxidation of light irradiation and ozone aging, implying that carbonaceous core oxidation caused worse health damage. Hence, it is necessary to consider the impact of widely used diesel exhaust treatment involving high temperatures, considering the potential toxicity of the resulting particles.