Technical Abstract: Ingestion of cold feed and water may suddenly reduce ruminal temperature, which could result in decreased microbial activity and diet digestibility. The objective of this study was to investigate the association between critical rumen in vitro incubation temperature and activity of ruminal microorganisms to produce gases and degrade NDF. Lyophilized ruminal extrusa (0.25 g) collected from ruminally cannulated cows grazing winter range in November 2010 (81.13% NDF, OMB) was weighed into thirty 100-ml glass syringes. Warmed McDougall’s buffer mixed 4:1 with rumen liquor donated by winter-grazing cows was added and syringes were placed in a 39°C water bath. After 12 h, syringes were randomly allocated to one of 3 water baths of different incubation temperatures, 39°C, 37°C or 35°C. These temperatures were selected based on previous findings that showed ruminal contents can drop intermittently below 35°C. Syringes were incubated for another 36 h. Rate and total gas production at 48 hours was reduced (P < 0.05) by lower incubation temperatures (rate: 0.63, 0.49, and 0.34 ± 0.01 mL/h; production: 29.3, 24.2, and 18.1 ± 0.51 mL/g of OM for 39°, 37° and 35°, respectively).Extent of NDF disappearance was reduced (P < 0.0001) by incubation temperature (21.7, 18.6 and 17.2 + 0.49% for 39°, 37° and 35°, respectively). Maximum gas production and NDF disappearance were found at 39°C. These data show the impact small differences in ruminal temperature due to cold water ingestion may have on rumen function.