Emptying the rumen of animals fitted with rumen cannulas is a regularly used experimental technique. This study in sheep aimed to evaluate whether total rumen emptying (TRE) results in disturbances in digestive parameters such as rumen pH, fermentation and microbial ecosystem, and whether it affects animal welfare. Eight adult wethers from two breeds offered hay and water ad libitum were rumen-emptied manually. Ruminal parameters were assessed 2 days before and 7 days after TRE. Welfare was assessed by measuring behavioural and cardiac responses to TRE involving human handling. Behavioural and cardiac measurements were performed (i) 2 h before, (ii) during and (iii) 2 h after the rumen emptying–filling session on wethers exposed to the same TRE operator. Results showed that TRE did not affect most of the digestive parameters analysed (ruminal pH, bacteria, methanogens) but did modify Dasytricha population and volatile fatty acid concentrations. Likewise, rumen emptying did not alter most behavioural and cardiac parameters, and we found no TRE-related change in fear reactions to the operator.