EEG studies have suggested that cattle perceive pain when bled without stunning. The present study on bleeding without stunning, compared cows that had received a local anaesthetic on the site of the bleeding cut (Lurocaine; Luro cows) one hour before bleeding with a 35 cm knife, with cows that had not (saline: Placebo cows). Various physiological indicators potentially related to pain or stress were evaluated. Increases in heart rate (P < 0.02) and cortisol levels (P < 0.001) during slaughter, compared to control levels are indicative of slaughter stress in both groups. GSH/GSSG ratio, plasma PGE2, TNFα, and NO levels and blood haematocrit levels at slaughter were not influenced by slaughter or treatment. At bleeding, excluding two out of the 15 cows with non-missing data, Placebo cows presented a longer delay between the loss of the corneal reflex and respiratory arrest. Post-mortem, Longissimus muscle of Placebo cows had a faster pH decline and remained warmer. Overall, results suggest greater stress levels in this group, probably due to pain. Plasma or brain β-endorphin contents in relevant brain structures did not increase following bleeding in either group, thus not supporting the hypothesis that stress or pain-induced release of endorphins reduces pain perceived following the cut. Furthermore, according to existing knowledge, plasma β-endorphins do not reduce pain perceived. Thus, both our study and previous research do not provide evidence that slaughter without stunning does not cause pain or other forms of stress in at least part of the animals.