The characteristics of the inoculum are recognized as one of the most relevant factors influencing the results of in vitro fermentations in batch cultures of ruminal microorganisms. Four rumen-fistulated sheep fed a 66:34 alfalfa hay:concentrate diet were used as donors to investigate the effect of rumen contents’ processing on in vitro methane (CH₄) and volatile fatty acid (VFA) production from 3 substrates of variable composition. Rumen contents were sampled from each individual sheep and subjected to the following treatments: SQ) squeezed through 4 layers of cheesecloth; FIL) SQ treatment and further filtration through a 100-µm nylon cloth; STO) treated with a Stomacher for 3 min at 230 rev min⁻¹ and followed by SQ. The resulting fluids were used as inoculum for batch cultures containing alfalfa hay, concentrate, or a 50:50 mixture of both feeds. Cultures were incubated at 39°C for 8 and 24 h, and CH₄ and VFA production was measured. There were no treatment × substrate interactions (P > 0.05) for any variable at any incubation time, excepting for the molar proportion of acetate at 24 h (P = 0.019). The method of processing the rumen contents did not affect (P > 0.05) either total VFA and CH₄ production or molar proportions of individual VFA at any time. At both incubation times, increasing the amount of concentrate in the substrate increased CH₄ production (P < 0.001, quadratic) and molar proportion of butyrate (P < 0.001, linear), but decreased acetate proportion (P < 0.001, quadratic) without affecting (P > 0.05) proportions of propionate. Whereas total VFA production was linearly decreased (P = 0.007) by increased amounts of concentrate in the substrate at 8 h of incubation, it was quadratically increased (P < 0.001) after 24 h of incubation. There were clear differences in CH₄ and VFA production among inocula from different sheep, which persisted across substrates. The results show that the tested methods of processing rumen contents did not affect in vitro fermentation characteristics of good quality substrates, but studies analyzing their possible influence on fermentation of low-quality substrates are required.