Efforts to characterize the organic matter (OM) properties in anaerobic digestates, comprising recalcitrant substrate (exogenous) and microbial-derived (endogenous) compounds, require further investigation. This study evaluated the identification of endogenous compounds during the anaerobic digestion (AD) of well-defined substrates with a subsequent starvation period. Substrates with different biodegradability levels and inocula from diverse origins were tested. Then, the digested OM was analyzed via ISBAMO fractionation and mid-infrared spectroscopy. The analysis of extracted fractions from the particulate OM of the inoculum revealed potential transfers of compounds to the dissolved OM after AD. The fluorescence complexity index of the dissolved OM fraction globally increased after AD and starvation periods when simple substrates were employed. Blank tests indicated that this increase was likely due to prolonged endogenous phases. Contrarily, partially biodegradable substrates accumulated slowly biodegradable protein-like and impeded the identification of soluble microbial products (SMP) via 3D fluorescence regardless of the inoculum type. Despite the systems fed with fully biodegradable substrates enabled the characterization of SMP, their profiles were influenced by multiple factors such as the substrate and inoculum type, as well as the digestion period. However, the impact of the selected substrates on the particulate OM was not detectable using Fourier transform infrared spectroscopy ratios or ISBAMO fractionation. Overall, the identification of endogenous compounds during anaerobic biodegradation was explored, contributing to a better understanding of the composition of digestates that could be valorized.