In vitro gas production techniques (IVGPT) are widely used to screen feeds and feed additives to reduce the number of animals needed for experiments, which in turn, reduces costs and increases animal welfare. However, information about repeatability is scarce. The objective of this study was to evaluate the variation from in vitro gas production fermentations in the same laboratory using the same feed substrate. The source of rumen fluid used in the fermentations was from two different farms with either cannulated lactating dairy cows or cannulated fasting heifers, representing two distinct stages of production (donor types). Seventeen 24 h fermentations, undertaken during a year, were used to evaluate the variation between the following parameters: gas curve parameters, baseline-corrected total gas production (TGP (mL at Standard Temperature and Pressure (STP))/g incubated dry matter (DM)), methane concentration (%) and yield (mL gas at STP/g DM), pH and degraded dry matter (dDM). Significant differences between donor types were found for the pH of the rumen fluid from individual animals and pH of fermented fluid. However, no significant differences were observed within donor type. The means for methane concentration and yield, after 24 h of fermentation, were not significantly different between or within donor types. Rate of early gas production was significantly different between donor types, but baseline-corrected TGP was not significantly different at 24 h. No dDM differences after 24 h of fermentation between or within donor types were detected. Gas production curves were different between donor types, being either a monophasic version of the sigmoidal model or an exponential curve for the heifers and the production animals, respectively. No differences were observed within type. Repeatability of rumen fluid (CV_RF), calculated as the coefficient of variation, and the associated parameters, which were investigated, was best for methane yield (CV_RFALL = 0.3%) and least for TGP at 3 h (CV_RFALL = 3%). Repeatability was dependent on donor type.