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Procedures for, and conditions during, inoculum collection and preparation, digestion, and residue recovery stages of the in vitro digestion technique were investigated to determine sources of within- and between-trial variability in digestion coefficients for forages of different chemical composition. Digestion coefficients differed significantly among inoculum preparation times of 1, 2, and 4 hours and for a decline in rumen fluid temperature to 29° in transport. These differences were not uniform across forage species and did not correlate with forage digestibility. Digestion coefficients differed significantly among inoculums prepared from fibrous deer rumen fluids that were strained only, strained and layered, and blended in a Waring blender and filtered through glass wool but did not differ between strained-layered and blended filtered inoculums of non-fibrous rumen fluid from a fistulated cow. Forage in vitro digestion in the absence of microbial activity (by solubility alone) indicated that forages having more soluble components were least affected by inoculums of different microbial activities, suggesting that between-trial differences be adjusted by a solubility, rather than a digestibility, factor. Inoculum nitrogen concentration did not correspond to between-trial differences in forage digestibility. Size of test tube, but not centrifugation versus filtration method of residue preparation, significantly affected digestion coefficients. However, because the standard large tube size cannot be centrifuged, the two methods of residue recovery would not be comparable unless the products of digestion were transferred from large tubes to centrifuge tubes. The end products of digestion must be stored under refrigeration if filtering proceeds for extended periods of time. This material was digitized as part of a cooperative project between the Society for Range Management and the University of Arizona Libraries. The Journal of Range Management archives are made available by the Society for Range Management and the University of Arizona Libraries. Contact lbry-journals@email.arizona.edu for further information. Migrated from OJS platform August 2020