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When animals are fed a grass and legume mixture, digestive interactions can occur in the rumen between the substrates contained in the different plants, and the response of the animal to the combination of forages can differ from the balanced median values of their components considered individually. Our objective was to assess the associative effects between temperate forages in 8 grass-legume binary combinations on in vitro rumen fermentation characteristics to highlight synergies or antagonisms in terms of nutritional and environmental impacts. Two grasses (cocksfoot, CF; ryegrass, RG) and 4 legume species (alfalfa, A; white clover, WC; red clover, RC; sainfoin, SAN) were incubated alone and in grass-legume mixture (1:1, wt/wt) in batches containing buffered rumen fluid during 3.5 and 24 h. For each substrate and each incubation time, parameters describing the degradation of the energetic and nitrogenous compounds and their partition into fermentation end products were measured. Data were subjected to ANOVA using a mixed procedure to test quadratic contrasts. At 3.5 h of incubation, many quadratic effects were observed. The presence of A, WC, or RC in mixtures quadratically increased the NH(3)-N production (up to +28% when compared with the calculated value, P < 0.001), whereas the presence of condensed tannin (CT) activity in SAN considerably decreased it (up to -67%, quadratic effect, P < 0.001) and the N disappearance. To a lesser extent, the presence of SAN reduced the proportion of methane in the gas produced (up to 7%, quadratic effect, P = 0.018). Generally, the degradation of OM and NDF was not stimulated by mixing grass and legume, except for SAN. However, the presence of SAN severely impaired the NDF digestion at the early phase of fermentation. At 24 h of incubation, few associative effects were observed in comparison with those observed at 3.5 h of incubation, but the effect of CT of SAN on N metabolism was still clearly present. This study shows that, among the grass-legume mixtures tested in vitro, only the presence of SAN can interact with the grasses to reduce the degradation of proteins and the production of methane but with a transitory negative effect on fiber digestion.