A mini dual flow fermenter, with a working volume of 80 mL compared to 1 L in previous devices, has been designed to study rumen microbial metabolism, potentially without the need of fresh inoculum. The study aimed to optimise a method of rumen content preservation to restore normal in vitro fermentations and methanogenesis after a 5-day adaptation period in the device. Different conditions (cryoprotectant, temperature of storage) and times of storage were tested. Two cannulated dry goats were used as donors of rumen inoculum. Each condition for each goat inoculum was tested in triplicate for 7 days with a diet (hay 0.6; barley 0.2, soybean meal 0.2) given twice a day at 0830 h and 1530 h. The first experiment tested the effect of freezing storage temperature (-20°C vs -80°C) with DMSO (5%) used as a cryoprotectant at 3 times of storage (fresh as control, 1 and 4 months). Daily gas volume and methane productions after 5 days of adaptation were similar in devices with fresh inoculum and with rumen inoculum stored at -20°C and -80°C for 1 month. After 4 months of storage, gas volume and methane production of inoculum stored at -80°C were closed to control ones but at -20°C methanogenesis was greatly impaired with a significant interaction between temperature and animal donor inoculum. The second experiment tested the use of 2 cryoprotectants (glycerol or DMSO) at 5%, at 3 times of storage at -80°C (fresh, 2.5 and 12 months). Gas volume and methane productions were closed to productions of fresh inoculum up to 12 months of storage. Even though gas volumes were higher with glycerol compared to DMSO, methanogenesis was similar with the 2 cryoprotectants. Based on these data, rumen inoculum can be kept for at least one year at -80°C without any gas production and methanogenesis impairment in our mini dual flow fermenters.