Anaerobic metabolism in a biological system, such as the rumen, depends largely on the reducing power of the bacterial population present. For that reason, it is crucial to understand the relationship between redox potential (Eh) and fermentative activity. The diverse microbial profile is characterized by enzyme systems capable of maintaining a constant, yet ever-evolving rumen environment. Variation in Eh is a result of intrinsic and extrinsic factors, including measurement procedure, diet, temperature, and animal. Because Eh is sensitive to oxygen contamination, the objective of this study was to assess different procedures for post-collection rumen inoculum to determine Eh. Four hours after feeding, ten rumen fistulated steers (267.9 8.4 kg), receiving a grower diet, had rumen inoculum (600 mL per animal) extracted using a vacuum suction device and Eh was measured in vivo simultaneously. One subset of samples was incubated in a 39C water bath (Procedure 1), while the second subset of samples was placed in an ice bath (Procedure 2). Redox potential and temperature were measured at 5, 15, 30, 60, 120, and 240 minutes for both Procedures. For Procedures 1 and 2, Eh values ranged from -235.4 to -313.1 mV and -198.0 to -293.4 mV, respectively. Data were analyzed using orthogonal contrasts with animal within-day and day as random effects in the statistical model. There was no effect for Procedure Time (P = 0.273) or Time (P = 0.396), but there were differences between Procedures (P > 0.001). Polynomial contrasts indicated quadratic tendencies for Procedure 1 and the interaction of Procedure x Time (P = 0.051 and P = 0.0776, respectively). This study concluded that Procedure 2 more accurately represents in vivo Eh values, likely due to the reduced rate of fermentation associated with samples preserved on ice.