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Non-lethal tissue sampling of insects is widely used to obtain DNA for ecological and conservation research, yet the potential for sub-lethal behavioural effects remains poorly understood. We experimentally evaluated three commonly used non-lethal sampling procedures in bumblebees—chilling, tarsal clipping, and whole-leg removal—in 160 workers from 15 Bombus terrestris colonies. Individual behaviour was recorded during foraging in semi-field flight cages and at colony entrances. Recovery time, foraging behaviours, and nest entry and exit behaviours were quantified. Chilling significantly increased recovery time relative to controls, but did not alter behavioural performance once activity resumed. Treatment did not affect the duration of individual foraging behaviours, including time spent on flowers, time between flowers, or visitation rate, nor did it influence nest entry, exit, or trip duration. Although less treated bees returned to forage than controls, there was no overall effect of treatment on the probability of returning to forage. However, there was a trend towards bees subjected to tissue removal making fewer foraging trips than chilled bees and controls while some activity-based measures, including proportion of time allocated to resting, differed among treatments. Together these results indicate that non-lethal sampling does not impair behavioural performance across most metrics, but that tissue removal may have some subtle impacts on overall activity levels and resting. While non-lethal methods remain preferable to lethal sampling, short-term behavioural effects may take place and should be further considered in future research to examine whether there are consequences for fitness.
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University College Dublin