Anaerobic co-digestion is a promising solution for nutrients balance and improvement of methane production in anaerobic digestion (AD) processes. However, the knowledge about the effects of different co-substrates in manure-based AD, and different feeding strategies, on the process performance and the methanogenic microbiome pathway, are still missing. Therefore, under harsh and slow stepwise increase of organic loading rate (OLR), by addition of lipids and carbohydrates as co-substrates in continuous reactors, this study elucidated their effect on methane production and methanogenic microbiome. The results showed that, when OLR increased by adding lipids, a severe inhibition due to accumulated long-chain fatty acids (LCFA) was observed, while no significant inhibition was obtained by addition of glucose. Additionally, the LCFA inhibition in the reactor fed with lipid was alleviated by slow stepwise feeding strategy that enriched aceticlastic Methanosarcina thermophile and Methanosaeta concilii, and hydrogenotrophic Methanobacterium methanogens.