Legislation governing non-stun slaughter of sheep in England requires that they are individually and mechanically restrained for slaughter and not moved for at least 20 s post neck cut, until unconsciousness or insensibility occurs. Complying with the need for individual handling, in what is a flock animal, has the potential to adversely affect welfare, in turn contravening the general legislative requirement to reduce any avoidable distress at slaughter. This study investigated the effects of individually loading and restraining lambs compared with the normal practice of group loading and restraint of lambs prior to slaughter when using a V-shaped restrainer. Rotating and static design loading pens were also compared to represent the range of conditions and facilities found across English abattoirs. Plasma cortisol and lactate concentrations were significantly lower in group-loaded animals and significant reductions were observed in the time duration of a range of components of handling as well as the average total time to load each lamb. Loading pen type had a less marked impact upon results, however, individual loading and restraint of lambs within a V-shaped restrainer appears particularly stressful for sheep in comparison with group loading. The loading pen type had a mixed effect although the rotating crowding pen is likely to have minimised physical exertion in lambs during loading and restraint. Based on these findings, group loading in a V-shaped restrainer, whilst complying with the 20-s standstill, is likely to be preferable in religious, non-stun slaughter of sheep.