As a popular method for to predict in vivo rumen function, in vitro fermentation has been utilized for many years. However, little is published regarding the ideal methodology used in in vitro systems. Furthermore, as new and improved technology is applied to in vitro rumen fermentation models, these methods need to be updated to improve model accuracy. The objective of this study was to identify the effects of altering the rumen fluid: substrate ratio on the outcomes of in vitro rumen fermentation. In this study, rumen fluid was collected from 3 ruminally cannulated, mature Holstein heifers. Buffered rumen fluid was incubated with either 0, 1, 1.2, 1.4, 1.6, 1.8, 2, 4, or 8 g dry, ground substrate. The fermentation units were kept at 39°C for 24 h, under constant agitation (60 rpm). Gas pressure and temperature were measured every 15 min using the Ankom Gas Monitoring System (Ankom, cat no #7056; Macedon, NY). Total gas production was calculated according to the ideal gas law. Fermentation was stopped by placing bottles on ice and a sample of rumen fluid was frozen for VFA analysis (HPLC) at Dairyland Laboratories Inc. (Arcadia, WI). Gas production was modeled using the Gompertz Gas model. Increasing the ratio of substrate to inoculum caused an increase in total VFA production, propionic, valeric, acetic, and butyric acids (P < 0.0001; all measured as mmol/L). pH decreased as the inclusion rate increased (P < 0.0001), as did the ratio of acetic to propionic acid (P < 0.0001) and production of NH₃ (mmol/L; P < 0.0001). The lag time (h) of fermentation was inversely correlated with the inclusion rate (P < 0.0001). No significant change was observed in the fractional rate of fermentation (mmol/ h); P > 0.05), and maximum gas production (mmol) was only significantly decreased in the 8.0 g treatment (P < 0.0001). It was established that between the ratios of 1.2, and 1.6 g dry substrate: 100 mL inoculum, none of the measured outcomes were statistically different (P < 0.05), and the environment of the fermenter units was comparable to that of the functioning rumen. Future studies should conserve this ratio to maximize quality of in vitro fermentation data before in vivo use.