AbstractBackgroundRumen juice analysis (RJA) involves analysis of a 10mL sample within minutes after sampling. However, it can be challenging to collect 10 mL of rumen juice (RJ) from some ruminants, and clinical circumstances can delay RJA.ObjectivesQuantify the effect of sample volume (2, 5, 10, 50, and 100 mL), and time‐to‐analysis (0, 30, and 60 minutes) on RJA.AnimalsCannulated cow.MethodsObservational experimental study. Two liters of RJ were collected at 26 separate times. The samples were subdivided into 2 duplicates of each sample volume at each sampling time; and analyzed at 0, 30, and 60 minutes after collection. Rumen juice analysis included pH measurement, methylene blue reduction time (MBRT), and protozoal motility.ResultsThe pH of 2 and 5 mL samples was significantly (P= .01) higher than the pH of 50 and 100 mL samples at all time points. The MBRT was significantly lower (faster bacterial reduction) for 100 mL samples compared to all other samples at 0 minute and to 2, 5, and 50 mL samples at 30 min. The pH and MBRT at 60 minutes were significantly higher than at 0 minute for all volumes (P< .05 andP< .01, respectively). For large protozoa, small sample volumes (2 and 5 mL) had significantly lower protozoal motility (scores of 5 and 4.5, respectively) compared to 100 mL samples at 60 minutes (score of 4;P< .05).Conclusions and Clinical ImportanceInterpretation of RJA could be affected by small sample volumes and delays to analysis. Sample volumes of ≥10 mL analyzed within 30 minutes after collection are recommended.