Pleistocene glacial stages were implemented into a 3D basin and petroleum systems model of the northeastern Netherlands to address the influence of low surface temperatures and the mechanical loading of ice sheets on the subsurface. Two ice sheet thickness scenarios were used based on published data. Overall, Quaternary glacial stages have a substantial impact on the temperature and pressure distribution in the subsurface. Subsurface temperatures are significantly reduced during glacial stages, leading to lowered present-day temperatures and a low geothermal gradient in the shallow subsurface. In deeply buried sedimentary formations, pressures build up with every glacial advance resulting in overpressures at the present day. Glacial stages do not directly influence the petroleum generation of petroleum source rocks in the area, but high pressures during loading might have impacted petroleum expulsion of the early mature Coevorden Formation. Hydrocarbon accumulations in the Lower Saxony Basin were simulated to investigate the possible effects of mechanical ice loading and unloading on hydrocarbon migration. A loss of Coevorden Formation-sourced hydrocarbons to the surface was calculated in the Lower Saxony Basin during the glacial stages, indicating an influence of glacial loading on the Mesozoic petroleum system.