This study examined the behavioural expression of cattle immediately prior to slaughter through the process of Qualitative Behavioural Assessment (QBA), and compared these results to measurements of physiology and temperament. Twenty-eight Angus steers were filmed while in a funnel chute as they were being moved towards an abattoir killing box. Footage of cattle was shown in random order to 15 observers. Observers assessed the cattle using a qualitative approach based on Free Choice Profiling (FCP) methodology, which gives observers complete freedom to choose their own descriptive terms. Data were analysed with Generalised Procrustes Analysis (GPA). There was significant consensus (P < 0.001) amongst observers in terms of their assessment of the behavioural expression of the cattle. Two main dimensions were identified, explaining 58.8% and 9.2% of the variation between animals, respectively. Low values for GPA dimension 1 were associated with terms such as ‘calm’ and ‘relaxed’ and high values associated with terms such as ‘nervous’ and ‘anxious’. Low values for GPA dimension 2 were associated with terms such as ‘annoyed’ and ‘frightened’ and high values associated with terms such as ‘curious’ and ‘interested’. We found no significant correlations between the QBA scores and three temperament measures (taken at weaning). However, cattle slaughtered towards the end of the line (rs = 0.45, P = 0.016) and cattle with a greater plasma lactate concentration (measured at exsanguinations) (R24 = 0.45, P = 0.020) were attributed higher GPA dimension 1 scores (i.e. more ‘nervous’/‘anxious’). The only animal with an ultimate muscle pH > 5.7 (classified as a ‘dark cutter’) had a low value for GPA dimension 2 (scored as relatively more ‘annoyed’/‘frightened’). The findings from this study suggest that QBA could contribute to assessing pre-slaughter animal handling, highlighting potential issues to be followed up with additional measures. The significant correlations between GPA dimension 1 with slaughter order and plasma lactate warrant further investigation, comparing behavioural expression with aspects of meat quality.