Inelastic neutron scattering (INS) is employed to examine the evolution of a promoter-free iron-based Fischer–Tropsch synthesis catalyst (∼10 g catalyst charge) that is exposed to ambient pressure CO hydrogenation at 623 K for up to 10 days time-on-stream (T-o-S). The longer reaction time is selected to better understand how the formation of a previously described hydrocarbonaceous overlayer corresponds to the catalyst conditioning process. Although the onset of pseudo steady-state reactor performance is observed at approximately 9 h T-o-S, INS establishes that the intensity of the C–H stretching mode of the sp3-hybridized component of the hydrocarbonaceous overlayer saturates at about 24 h T-o-S, while the corresponding intensity of the C–H stretching mode of the sp2-hybridized component requires 100–200 h T-o-S to achieve saturation. This novel series of measurements reveal different aspects of the complex catalyst evolutionary process to be indirectly connected with catalytic turnover.