This in vitro experiment used a 4 × 2 × 2 factorial design with 6 replicates (3 per run) to determine effects of rumen fluid treatment, substrate and probiotics. There were 4 treatments of rumen fluid inoculant, 2 pre-weighed substrates (0.5 g timothy hay or 0.5 g timothy hay with 0.5 g corn grain), each with or without a probiotic supplement containing yeast, lactic acid bacteria, and digestive enzymes. Control (C) rumen fluid (10 mL/flask) was collected 4 h after feeding, and was blended and strained. Other rumen fluid treatments used 0.5 mL rumen fluid: otherwise as control (A), collected before feeding (B), or not blended (D). Samples in 40 mL of medium plus inoculant were incubated at 39°C for 24 h. Results were analyzed by the model: Y = μ + T + S + P + R + all interactions + E, where Y is the response variable, and T, S, and P are fixed effects of rumen fluid, substrate, and probiotic, and R and E are random effects of run and residual error. Differences were reported at P < 0.05. NDF digestibility at 24 h was lower for the treatments with corn (residual NDF 66.2%) compared with treatments with hay alone (residual NDF 57.8%). NDF digestibility was greater for C, and for C and B for treatments without corn. Total gas production in mL per g digested OM was higher for C (316 mL/g) and B (279 mL/g) than A and D (241.5 mL/g). Total VFA were highest for B with corn (178 vs. 171–181 mM), and lowest for D without corn (75 vs. 82–88 mM). Acetate concentration increased most in treatment C with corn (91 mM), compared with other treatments with corn (74–76 mM) and treatments without corn (43–51 mM). Propionate concentration increased most in treatments with low volume of inoculant with corn (91–95 mM), compared with C with corn (38 mM), and without corn (17–22 mM). Butyrate concentration was greatest for treatment C with corn (24.1 mM), compared with other treatments with corn (9.3–11.6 mM), and without corn (4.9–6.9 mM). Probiotic effects were not consistent across runs, but were exhibited within runs for digestion, VFA and gas. Ethanol was detected in low-volume inoculants with corn (9.8–14.7 mM). Low microbial competition shifted in vitro fermentation toward propionate.