The effects of short-term road transport on dairy cow welfare have been the focus of very few studies. This study is the first to evaluate whether short-term transport of dairy cows under conditions similar to a typical journey from farm to slaughterhouse can cause nonlame cows to become lame. In total, 203 Danish Holstein cows from 18 different commercial Danish dairy herds were included in the study. In each herd, a large number of cows were locomotion scored and 8-12 non-lame cows were selected for transport. After the initial locomotion scoring, the selected cows were loaded onto a truck and transported on average 84 km (1 h 55 min) using a route simulating a typical transportation of cows to slaughter under Danish conditions. Returning to the herd of origin, the cows were immediately unloaded and locomotion scored by the same observer as prior to transport. Results showed that locomotion score did not change significantly and that no cows became lame as a consequence of the transport.