Introduction: Batch cultures are commonly used to study ruminal digestion because they are easy to implement. Nevertheless, most are short term studies (a few hours) that are unable to evaluate effects of dietary changes that affect the microbiota, which needs more than 24h to become apparent. So, this study aimed at evaluating persistency of microbial activities during medium-term (96h) cultures of ruminal contents. Animals, material and methods: Ruminal contents were taken from two rumen-fistulated dairy cows receiving a meadow hay based diet (58% NDF and 3% starch). One portion of the contents was used for an immediate measurement of microbial activities (disappearance of fibre, starch and nitrogen, and biohydrogenation of unsaturated fatty acids) and the second portion was incubated during 96h, with a renewal of half of the 200mL media every 12 hours with substrate and 7-pH bicarbonate buffer. This incubation simulated the rumen of a cow receiving 2 equal meals per day, containing 50% wheat bran + 10% soybeans + 40% meadow hay (51% NDF and 5% starch), and having a 6% per hour ruminal emptying, on average. Fermentation parameters (volatile fatty acids, VFA; ammonia, NH3; pH) were recorded every 12h. After 96h, microbial activities of the cultures were assayed as in the fresh rumen content during the first day. The results obtained with fresh ruminal contents on day 1 were compared to those obtained with cultures, using GLM of SYSTAT. Results and discussion: Fermentative activities were maintained during cultures: pH and total VFA concentrations remained constant. Nevertheless a decrease of acetic acid proportion (C2) and an increase of propionic acid proportion (C3) were observed, with a reduction from 3.9 to 2.7 of the C2/C3 ratio, associated with a 22% increase of butyric acid (C4) proportion. NH3 concentration decreased by about half during the first 48h and remained stable thereafter. Compared to fresh ruminal contents, 96h cultures possessed a twice lower fibre disappearance, a similar nitrogen disappearance, a twice higher starch disappearance and a 24% higher biohydrogenation extent of linoleic acid with an increase by 61% of trans-11 isomers. Nitrogen degradation was not modified by medium-term cultures, and the lower NH3 concentration could have resulted from volatilization of aqueous ammonium solution (boiling temperature: 38°C) during our 39°C incubations. Fibrolytic activity and consequently C2 concentration were not favoured by the mid-term cultures, contrary to amylolytic activity and its associated C3 concentration, most probably because of particle size of culture substrates which had been finely ground. The increase of C4 concentration was in agreement with the increase of linoleic acid biohydrogenation by trans-11 pathway, since this pathway would be mainly due to Butyrivibrio fibrisolvens (Maia et al., 2007), a fibrolytic bacteria producing C4 and preferring hemicelluloses (Dehority, 2003), which was provided by the wheat bran in our experiment Conclusion: This incubation procedure can be used to compare medium-term effects of dietary treatments on rumen microbial digestion in cultures. Such cultures do not allow a quantitative measurement of the effects but allow screening procedures and description of the nature of the effects. Further studies will investigate changes of microbiota using pyrosequencing.