Current ruminant feeding systems depend on knowledge of the composition of feeds and the rate and extent of degradation of feed organic matter (OM) and crude protein (CP) in the rumen. The effect of storage and preparation of samples on in vitro gas production and fermentation characteristics of two common forage species, namely alfalfa and rye grass were studied. Samples were prepared as fresh (F), frozen-thawed (FT) and FT + starch (FT + S) before in vitro evaluation. The fractional rate of loss of organic matter (OM) and the total N and total VFA production during 12h of incubation were significantly faster for alfalfa than for rye grass. Model parameters describing changes in OM loss and total N appearance differed significantly between F samples and FT and FT + S samples; there was a significant interaction between forage species and preparation method for fractional degradation rate of total N. A significant interaction between forage species and preparation method at 6 h incubation changed the rankings. The propionate:acetate ratios after 12 h incubation were similar for alfalfa and rye grass but were lower for F and FT samples than for FT + S samples. After 12 h of incubation, alfalfa produced more gas, total VFA (mmol/g OM) and microbial crude protein (mg/g OM) than rye grass, whereas F samples produced more fermentation products than FT and FT + S samples. In vitro degradation characteristics of forage samples were influenced by forage species, but also by sample preparation method; therefore, consistent use of one sample preparation method is recommended when comparing degradation characteristics of forage species in vitro.