In vitro methods have been developed to measure digestibility, but such methods may not accurately reflect gas production or volatile fatty acid (VFA) profiles. The objective of this study was to determine the effect of different in vitro conditions on VFA and gas production. The experimental design was a 4 × 2 × 2 factorial CRD with four replicates. Treatments were four ratios of medium to rumen fluid by volume (5:95, 25:75, 50:50, and 75:25), two concentrations (w/v) of added timothy hay (0.5% or 1%), with or without added sodium acetate (increased initial concentration by 50 mM). Total volume of medium and rumen fluid was 10 mL per tube. Measurements of gas production and VFA were recorded at 0, 4, 16, 24, and 48 h. Statistical analyses used a mixed model including all fixed effects and interactions with tube as a random effect, and time nested within tube. Total gas production increased (P < 0.001) with higher medium proportion. The final pH increased (P < 0.0001) as medium proportion increased. Medium proportion positively affected (P < 0.05) overall average concentration of both acetate production and propionate production. Higher hay concentration increased (P < 0.0001) total gas produced from 0 to 48 h, increased total acetate production (P < 0.01), propionate production (P < 0.001), and decreased pH between 24 and 48 h (P < 0.0001). Sodium acetate addition increased (P < 0.0001) pH between 24 and 48 h. Acetate:propionate (A:P) concentration decreased over time (P < 0.0001). Initial rumen fluid A:P ratio was 3.7 but average A:P ratio of produced VFA started at 2.2 and increased to 2.50 (SE = ±0.51). The A:P ratio differed for VFA produced in vitro compared to initial rumen fluid, but no tested treatments were found to change A:P ratio.