Residual biomass shows potential to be used as a feedstock for fast pyrolysis bio-oil production for energetic and chemical use. Although environmentally advantageous, further catalytic upgrading is required in order to increase the bio-oil stability, by reducing reactive compounds, functional oxygen-containing groups and water content. However, bio-oils may separate in fractions either spontaneously after ageing or by fractionated condensation. Therefore the effects of upgrading on different fast pyrolysis bio-oil (FPBO) fractions obtained from a commercially available FPBO were studied by elemental analysis, GC-MS and 1H-NMR. Not only the FPBO was upgraded by catalytic hydrotreatment, but also the heavy phase fraction formed after intentional aging and phase separation. The reactions were conducted between 175 and 325 °C and 80–100 bar by using a nickel–chromium catalyst in batch experiments. The influence of the hydrotreatment conditions correlated with the composition of the upgraded products. Higher oxygen removal was obtained at higher temperatures, whereas higher pressures resulted in higher hydrogen consumption with no significant influence on deoxygenation. ... mehrAt 325 °C and 80 bar 42% of the oxygen content was removed from the FPBO. Compounds attributed to pyrolysis oil instability, such as ketones and furfural were completely converted while the number of alcohols detected in the upgraded products increased. Coke formation was observed after all reactions, especially for the reaction with the fraction rich in lignin derivatives, likely formed by polymerization of phenolic compounds mainly concentrated in this phase. Independently of the feedstock used, the upgraded bio-oils were very similar in composition, with reduced oxygen and water content, higher energy density and higher carbon content.