The objective of this study was to determine the effect of time of rumen fluid collection relative to feeding on gas production kinetics and volatile fatty acid (VFA) production for in vitro rumen fermentation. Three ruminally cannulated Holstein heifers were rumen fluid donors. Feed was removed from heifers 12 h before feeding, rumen fluid was collected from each heifer before feeding (0 h), and at 2, 4, and 6 h after feeding, repeated on 3 separate incubation days. Buffered rumen fluid (100 mL) was incubated in 250-mL bottles containing 1.4 g of dried TMR, in duplicate for each heifer at each collection time. All bottles were incubated for 24 h at 39°C and constant agitation (60 rpm), and capped with monitors to capture temperature and pressure every 15 min (RF1, Ankom Technology, Macedon, NY). Gas data were fit with nonlinear regression in GraphPad Prism 7 using the formula y = V_m(1−(e^(-K_d^(x-lag)))) where y is gas produced at time X (mmol), V_m is the asymptotic gas production (mmol), K_d indicates the fractional rate of gas production (mmol/h), X is time (h), lag refers to the lag time before the start of fermentation (h). At the end of incubation, final pH and a sample of rumen fluid was collected for VFA and ammonia nitrogen. Data were analyzed using PROC GLIMMIX of SAS, with donor as the experimental unit and day as the random blocking factor; significance defined as P ≤ 0.05. Time of rumen fluid collection significantly affected gas production kinetics (lag P = 0.01, V_mP = 0.03, K_dP < 0.0001), acetate (mmol/L; P = 0.0004), propionate (mmol/L; P = 0.02), isobutyrate (mmol/L; P < 0.0001), valerate (mmol/L; P = 0.004), isovalerate (mmol/L; P < 0.0001), and total VFA concentrations (mmol/L; P = 0.004). All VFA relative proportions were altered due to time of rumen fluid collection (P < 0.02). Gas production was highest in fermentor units fed with rumen fluid collected 2 h post-feeding and VFA production was highest when rumen fluid was collected 4 h post-feeding. Our findings suggest gas and VFA production are maximized when rumen fluid is collected between 2 and 4 h post-feeding.