Behavioural analysis has an important role in the assessment of stress in animals. Fractal analysis provides a novel measure of behavioural complexity and has previously revealed subtle alterations in behaviour under biologically costly conditions, such as parasitism or disease. The analysis is based upon the temporal pattern of behaviour that, although rarely considered in behavioural studies, may provide information in addition to standard measures of duration and frequency. Such information could be useful in assessing the welfare of confined animals. The objective of this experiment was to test the hypothesis that fractal analysis reveals novel behavioural alterations during stress using ISA brown pullets. The behaviour of undisturbed birds in their home pen was compared to the behaviour of the same birds: (1) in a novel arena, (2) in their home pen following blood withdrawal and (3) in their home pen following 5 min of mechanical restraint plus blood withdrawal. Detrended fluctuation analysis (DFA) was applied to sequences of vigilance behaviour and walking. In summary, this study concluded that DFA provides a novel measure of temporal behavioural complexity in chickens. Acute stress caused an increase in behavioural complexity in the present experiment suggesting that DFA can reveal more subtle changes in behavioural organization during stress. If such behavioural alterations represent a non-specific stress response this methodology could allow objective comparisons of different stressors to be made.