Consumer concerns about the welfare of laying hens are increasing, leading to increased interest in identifying reliable ways to assess welfare. The present study evaluated invasive and non-invasive welfare indicators in relation to a stressful challenge. The study included 126 Lohmann Selected Leghorn hens housed in furnished cages. Welfare indicators were measured between 61 and 70 wk of age in birds excluded from their nests for 5 consecutive d and control birds that had continuous access to nests. Baseline recordings were carried out in both groups prior to and post exclusion period. The assessed indicators were: corticosterone metabolites in droppings (FCM), corticosterone concentration in yolk, corticosterone concentration in plasma, irregularities of eggshells, heterophil to lymphocyte (H:L) ratio, tonic immobility duration, and feather cover. Behavioral observations showed that the birds had a clear preference for using the secluded nest sites, confirming that they were likely to perceive nest exclusion as an undesirable experience. Further, elevated levels of FCM in droppings, yolk corticosterone concentrations, H:L ratios and irregular eggshells were detected in both nest deprived and control birds during the exclusion. This suggests that these indicators were able to detect an increased stress response arising from nest deprivation, and it is hypothesized that the stress spread to birds in adjacent cages with access to nests. There was a positive and consistent correlation between FCM in droppings and eggshell irregularities, also supporting the use of eggshell irregularities as a potential non-invasive welfare indicator. However, the pattern of the stress response varied between indicators and correlations were generally few and inconsistent, highlighting the complexity of the relationship among welfare indicators.