A single-cylinder engine test bench is recently built at Biomass Technology Group B.V. (BTG). In this report, experimental studies are carried out to investigate engine performance using Fast Pyrolysis Bio-Oil (FPBO). Diesel and FPBO+E10 (FPBO blended with 10 wt% ethanol) are tested at a wide range of operating conditions. Fuel consumption and exhaust emission are measured. Based on the needle lift, fuel pressure, and cylinder pressure data, a set of indicators are defined to evaluate fuel injection and combustion characteristics. Results show that for the same settings of the mechanical inline pump, FPBO+E10 shows an almost doubled injection duration, slightly advanced injection timing, and higher injection pressure than diesel. The autoignition of FPBO+E10 requires a high in-cylinder temperature, e.g., by heating the intake air temperature to 125 ºC or higher. The ignition delay of FPBO+E10 is close to that of diesel, while the burn duration of FPBO+E10 is much shorter. For the single-cylinder engine running on FPBO+E10, the optimized engine net indicated efficiency is 53.6%, and the indicated specific fuel consumption is 373 g/kW·h. However, the maximum brake efficiency is just 21.8% due to parasitic losses. Generally, FPBO+E10 has comparable emission levels with diesel. At the intake temperature of 125 ºC, FPBO+E10 has slightly higher CO and HC emissions but around half the NOx emissions compared to diesel operation.