Eight groups of six red deer (four groups of males and four groups of females) were held overnight in an unfamiliar paddock, after which they were moved into a holding pen and slaughtered sequentially by stunning with a captive bolt pistol in a restraining pen. On entering the restraining pen, half the deer in each group were stunned immediately, but the other half were stunned after 10 minutes. Deer spent much of the initial period in the holding pen standing stationary in ‘alert’ postures, but became less alert over time. A significantly higher proportion of males than females were observed in alert ‘head-up’ postures in the restraining pen. Deer held in the restraining pen for 10 minutes had significantly higher plasma cortisol concentrations than those which were stunned immediately. Females had a significantly higher glycogen concentration postmortem in both the liver and semimembranosus muscle than males, and deer held in the restraining pen for 10 minutes had a significantly lower liver glycogen concentration than deer which were slaughtered immediately. Males had significantly higher semimembranosus muscle pHu. values than females, although the differences were small, and of the 14 deer with pHu. values above 6.0, 11 were males.