The carbon dioxide emissions into the atmosphere by the transport sector demands an urgent reduction, this implies the necessity to use alternative fuels such as biodiesel (FAME-Fatty Acid Methyl Ester) and HVO (Hy-drotreated Vegetable Oil) in Diesel engines. One of the advantages of both these fuels is the compatibility with current and future vehicle fleets. To investigate the possible effects of using these alternative fuels in Heavy-Duty engines, a methodology was defined, and an experimental set-up was prepared to identify the blending influence on the operation of an engine. In the present study, 6 different blends were considered containing different ratios of HVO, commercial diesel and FAME. A heavy-duty vehicle (HDV) was used in a chassis dynamometer considering performance tests and fuel consumption in 11 stabilized points that were usually common in regular vehicle use. The engine performance and fuel consumption results revealed the potential and weaknesses of using these fuels compared to traditional fuel, revealing the possibility of reducing CO2 emissions due to the renewability origin, with the highlight that the use of HVO and reduced FAME blends showed no loss of engine performance, but a slight reduction in torque was detected when using HVOxBx and BxHVOxD.