Physiological and behavioural measures of stress in caged hens on a commercial farm were compared between White Leghorn and brown Hyline strains, and between three tiers of cages. Blood and faecal samples were collected from undisturbed birds for corticosterone measurements. Plasma corticosterone responses to a stressor were measured by the collection of blood samples after 15, 30, and 60 min of a handling stressor. Tonic immobility and novel object tests were used to measure fear behaviour. Plasma corticosterone in undisturbed hens and faecal corticosterone did not differ between White Leghorn and brown Hyline hens, whereas the plasma corticosterone response to a handling stressor was greater in White Leghorns. The duration of tonic immobility, latency to first head movement and number of head movements in tonic immobility tests were greater in white than brown birds, whereas the number of inductions was less for tonic immobility tests. There were no differences between the strains in their responses to a novel object. There were no differences between tiers in plasma corticosterone or corticosterone responses or tonic immobility responses, and no consistent differences in responses of birds to a novel object. This is the first study in which plasma and faecal corticosterone concentrations and fear behaviour have been measured together in laying hens, and the first description of plasma corticosterone responses to handling over 60 min for caged laying hens on a commercial farm. The study has shown the value of measuring endocrine and behavioural variables together to provide objective data on characteristics of different strains of hens.