Sampling hour matters: diel variation in leukocyte profile of red‐legged partridges

Leukocyte counts are widely used as integrative biomarkers of immune function and physiological condition across ecological and biomedical sciences. However, their reliability can be compromised by sources of variation related to practical issues associated with sampling. Here, we investigated whether leukocyte profiles were affected by the hour of sampling in adult captive male red‐legged partridges Alectoris rufa . We used a repeated‐measures design in which birds were sampled once per week over four consecutive weeks in May. In each weekly sampling session, individuals were sampled at one of four predefined time points across the day (06:00 h – before sunrise –, 10:30, 15:30, 20:00 h, GMT+1). Total leukocyte (WBC) counts and the absolute numbers of all leukocyte types showed pronounced daily fluctuations, with minima at mid‐morning and recovery by afternoon or evening. By contrast, relative leukocyte proportions and the H:L ratio were more stable, although eosinophil and monocyte proportions varied significantly across the day. Effect sizes indicated that the strongest diel variation occurred in total WBC counts and the absolute counts of lymphocytes and monocytes. In addition to diel patterns, we detected mainly proportional leukocyte variation across sampling days. These fluctuations likely reflect transient physiological or environmental factors, although their cause remains uncertain. Handling time also significantly affected most leukocyte variables, with longer handling intervals producing rapid changes in the number and proportion of leukocyte types. To minimise potential temporal bias in leukocyte measurements or facilitate its statistical control, we recommend standardising sampling within narrow time windows, recording the sampling hour, and minimizing time elapsed between capture and sampling.