Properties of alternative fuels, such as cetane number, aromatic content, bulk modulus, etc., can vary significantly; thus, a detailed investigation is conducted to assess the impacts on engine combustion, efficiency, and emissions during steady-state and transient engine conditions. Insights can be used to maximize the benefits of alternative fuels or to avoid potential problems that stem from differing ignition and combustion characteristics of particular fuels. In this work, biodiesel (20%, 50%, and 100% biodiesel blends), jet fuel JP8, and synthetic jet fuel S8 are compared to diesel #2 (ULSD). Comparisons are made on ignition, combustion, particulate matter, particulate size spectra, and NOX emission at steady state under harmonized operating conditions, that is, with equalized fuel energy input. All experiments are conducted using the same baseline calibration originally developed for diesel fuel. Ignition delay and combustion phasing trends suggest the need for possible adaptation of ECU calibration. The impact of alternative fuels on particulate emission depended strongly on fuel aromatic content and oxygenation. NOX emission was correlated to multiple physical and chemical fuel properties. While the significance of different properties on NOX emission varied with engine condition, the CA50 location was strongly correlated at all conditions. This paper also includes detailed insight into particulate spectra obtained for all six fuels. Transient engine operation over a driving schedule was characterized with the engine-in-the-loop setup. The use of alternative fuels caused more aggressive cyber-driver behavior as a reaction to their lower energy densities. Different fueling histories were recorded with the alternative fuels and led to marked changes in instantaneous emissions traces. In particular, spikes of particulate emission in the exhaust that typically occur at accelerator tip-in were reduced with biodiesel, while extended high load operation was observed in order to follow the demanded vehicle velocity trace and led to higher NO. Cumulative results over the complete FTP 75 driving schedule indicate that transient engine operation reduces the particulate matter benefits of the alternative fuels. JP8 and S8 show slight NO emission benefits with biodiesel showing slightly worse NO emission when compared to steady state. The more aggressive driver behavior led to worse fuel economy.