How important are sampling and experimental design decisions in shaping test subject behaviour under laboratory conditions? We examined the effects of circatidal rhythm, time held in captivity, sampling location (open or covered areas of habitat), acclimation period and water depth on activity and emergence latency in hermit crabs (Pagurus bernhardus). We found that subjects held in captivity for 1 month and those collected from the open were faster to emerge from their shells after disturbance compared, respectively, to those tested after 1 day in captivity or collected from beneath cover. We also found that subjects tested after shorter acclimation periods were more active than those tested following longer acclimation periods. Our findings reveal that sampling and study design decisions can have pronounced influences on subject behaviour measured under otherwise common conditions, with potentially important implications for interpretation and reproducibility of findings. As researchers we should take care to explicitly consider how sampling biases and effects arising from our experimental protocols might affect the behavioural responses of test subjects. Doing so can help us make more reasonable generalizations beyond our subject pool, draw better-informed comparisons between studies and achieve greater reproducibility of findings.