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Aim of this paper was to assess the feasibility of the application of wood pyrolysis oil (WPO) as a fuel for medium-duty Diesel engines. The experimental activity was carried out both on a diesel injection system and on a DI Diesel engine. High-speed visualization was used to highlight the spray characteristics and an instrumented test bench to evaluate engine performance and emissions. No modification was carried out on the engine and the efforts were addressed to make the WPO compatible with engine operation. Accordingly, WPO was not tested as a pure fuel, but in blends with diglyme and in emulsions with Diesel fuel.