Bitumen from crude oil refinery is an important construction material as the binding component for asphalt mixes in road construction. Due to its organic composition, it is susceptible to oxidation when being exposed to reactive oxygen species (ROS). Oxidation is the main factor driving long-term ageing of the bitumen, which leads to an increase of brittleness and stiffness at lower temperatures and thus, makes road constructions more prone to cracking. Ageing is therefore a limiting factor for the service life of road infrastructure. In the presented study, the impact of different ROS on oxidative ageing of bitumen is studied. Asphalt mix specimens are aged in a specially designed device (VAPro) by leading different gases through the specimen, i.e. pressurized air, O-2, O-3 enriched O-2 and dried air enriched with O-3 and NOx. The flow rate is 1.0 L/min and the conditioning time is set to 7 days for air and 3 days for all other gases, respectively. Temperature (+60 degrees C) and pressure are set to simulate realistic field conditions. Bitumen is extracted and recovered from the specimens after ageing and assessed rheologically by DSR analysis. It was found that neither ambient atmosphere, nor pure O-2 or O-3 enriched O-2 by themselves induce relevant oxidative ageing. By combining air, O-3 and NOx, high oxidative ageing levels comparable to RTFOT + PAV can be achieved. Thus, realistic, yet efficient laboratory ageing methods for bitumen and asphalt mixtures need to take all ROS, especially NOx, into account. (C) 2020 The Author(s). Published by Elsevier Ltd.