In vitro systems involving microbial fermentation such as the Hohenheim gas test (HGT) typically need freshly obtained inocula such as rumen fluid or faeces. The objective of this study was to test the possibility of becoming independent of fresh faeces by using preserved faeces. Fresh faeces from sheep (control, C) was compared with seven differently preserved faeces in the HGT by using nine feeds and studying in vitro gas production (GP). Preservation involved freezing at -20°C (FR), shock-freezing with liquid nitrogen (N) and additional freezing at -20°C (FRN), FRN followed by defrosting (FRNdef), shock-freezing with liquid N and freeze-drying (FDN), freeze-drying (FD), freeze-drying and storage for three weeks (FD3W) and six months (FD6M). MA metaproteomics workflow was used to analyse the effects of treatments C, FR, FRN, FD, and FDN on to study changes in microbial community composition and functionactivity.