The development of in vitro methods to accurately estimate gas production and volatile fatty acid (VFA) profile in rumen fermentation would enable isolation of fermentation effects from animal interactions. This experiment compared 4 headspace gas combinations with or without addition of 50 mM sodium acetate. Gas headspace treatments were: 1) CO2(100%), 2) CO2–CH4(50/50), 3) CO2–H2(95/5), and 4) CO2–CH4–H2(47.5/47.5/5). Each treatment was replicated in 4 tubes with repeated measures of VFA and gas volume taken at 0, 4, 16, 24, and 48 h. Timothy hay (0.1 g) was added to 20-mL tubes, and 0.5 mL sodium acetate solution or buffered medium were added to each tube. Tubes were equilibrated with each gas mixture before adding 9.5 mL rumen fluid. Tubes were incubated at 39°C while shaking with 20-mL syringes attached to collect and measure produced gases. Butyrate production at 4 h was affected (P< 0.05) by gas composition, and was: 2.96, 3.09, 2.33, and 1.44 (mM; SE ± 0.437) for treatments 1–4. Propionate production at 48 h was affected (P< 0.05) by gas composition, and was: 8.71, 8.97, 10.60, and 7.12 (mM; SE ± 0.789) for treatments 1–4. Gas production at 4 h was 1.08, 2.70, 0.98, and 1.43 (ml, SE ± 0.327) for treatments 1–4. Lower starting concentration of CO2in headspace gas may have caused CO2efflux from the buffer. There was a trend (P< 0.1) for an effect of the gas mix at 24 h on the acetate: butyrate (A:B) ratio of produced VFA. A:B ratios of produced gases were: 2.95, 2.77, 2.87, and 2.18 (mM; SE ± 0.208) for treatments 1–4. In contrast to expectation, there was a trend (P< 0.08) for greater acetate production with acetate addition (10.71 mM) than without (7.11 mM, SE ± 1.413). Initial gas composition of in vitro procedures can affect gas production and VFA profiles with higher percentage of CH4and H2in headspace (more reduced conditions) favoring propionate and butyrate over acetate and gas production.