Sampling and analyses of ruminal contents are important tools in ruminant nutrition research. The use of rumen-cannulated cows is the standard method to obtain representative samples of ruminal contents, but the use of an oral stomach tube can be a non-invasive option to collect rumen samples in intact animals. The objective of this study was to compare rumen fluid samples collected through a rumen cannula (CS) or using an oral stomach tube (ST) for measurement of pH, protozoa counts, dissolved hydrogen and volatile fatty acids (VFA). Six rumen-cannulated lactating Holstein cows were fed a standard diet for an adaptation period of 2 wks followed by sampling during the third experimental week. Rumen samples were collected at 0, 2, 4, 6, 8, and 12 h after the morning feeding in 2 consecutive days. CS samples were taken from the ventral sac, the atrium/reticulum, and 2 samples from the feed mat; composited samples were filtered through 2 layers of cheesecloth for further analyses. In parallel, ST samples were collected by inserting the stomach tube via the esophagus into the rumen to an approximately 120 to 150 cm depth. The first 100 to 200 mL of the ST rumen fluid samples were discarded. Data were analyzed using the MIXED procedure of SAS including treatment, sampling time, and their interaction in the model; cow was random effect. Compared with CS, ST samples had or tended to have greater (P < 0.01) pH (6.26 and 6.73, respectively; SEM = 0.13), lower (P < 0.01) protozoal counts (26.7 and 8.70 × 10⁴, respectively; SEM = 5.10 × 10⁴), and lower total (P < 0.01; 142 and 109 mM, respectively; SEM = 5.44) and individual VFA concentrations (P < 0.01), except for isobutyrate (P = 0.30). Molar proportions of individual VFA tended to be lower (P ≤ 0.07) for ST compared with CS, except for butyrate and propionate (P ≥ 0.17). There was no difference (P = 0.51) in dissolved hydrogen concentration between sampling methods. In conclusion, rumen fluid samples collected through an oral stomach tube are not representative of protozoa counts and fermentation variables as measured in samples of ruminal fluid collected through the rumen cannula.