Interassay error caused by the inconsistent nature of rumen fluid inoculum confounds comparisons of forage in vitro neutral detergent fiber (NDF) digestibility (NDFD) analyzed in different repetitions or laboratories. Our objective was to determine if priming rumen fluid and allowing it to produce a standard amount of gas before inoculating samples improved assay repeatability. In 2 experiments, we compared interassay error of NDFD estimates between several in vitro assays. In both experiments, dried, ground (1 mm) alfalfa samples (0.5 g) sealed in bags were placed in 125-mL Erlenmeyer flasks and incubated with in vitro media and 10 mL of rumen fluid. In experiment A, rumen fluid was collected from a cannulated cow fed a high forage diet and prepared one of 2 ways; rumen fluid was either used immediately after it was collected and strained through cheese cloth (GVA), or strained fluid was combined with buffer, reducing solution, and 12.5 mg of cellulose/mL of rumen fluid and allowed to produce a consistent amount of gas before inoculation (RPA). The assay was repeated 5 times, with 13 samples per method. In experiment B, inoculum was prepared one of 3 ways; RPA, GVA except rumen fluid was collected and pooled from 2 cows (GVB), or RPA with fluid pooled from 2 cows. The in vitro assays were repeated 5 times with 8 samples per method. Neutral detergent fiber was analyzed using a forage fiber analyzer and 24-h NDFD was determined as: NDFD (% of NDF) = 100 x [(NDF(0h) - NDF(residue))/(NDF(0h))]. Data for each experiment were analyzed using a mixed model procedure and repetition sum of squares for each technique was determined and compared with an F-test to assess technique interassay error. In both experiments, rumen fluid priming significantly reduced repetition sums of squares, 51.2 versus 503 and 23.3 versus 164, compared with the respective GVA or GVB. However, priming significantly decreased NDFD values, 22.5 versus 24.8 and 23.9 versus 26.6%, compared with GVA and GVB, respectively. Priming rumen fluid with cellulose improved in vitro NDFD assay precision, but depressed in vitro NDFD.