Laboratory mice are the most frequently used animals in biomedical research. In accordance with guidelines for humane handling, several blood sampling techniques have been established. While the effects of these procedures on blood quality and histological alterations at the sampling site are well studied, their impact on the animals' welfare has not been extensively investigated. Therefore, our study aimed to compare three commonly used blood sampling techniques regarding their effects on different indicators of animal welfare, including physiological and behavioural response stress parameters, including pain measures, home-cage behaviour and nest-building as well as exploratory activity and neophobia. Male C57BL/6J mice were subjected to a single blood collection from either the vena facialis, the retrobulbar sinus or the tail vessel, or were allocated to the respective control treatment. While all blood sampling techniques led to an acute increase in plasma corticosterone levels, the response was strongest in animals that underwent sampling from the vena facialis and the retrobulbar sinus. Similar results were observed when the time-course of adrenocortical activity was monitored via corticosterone metabolites from faecal samples. Blood collection from the vena facialis and the retrobulbar sinus also decreased exploration of novel stimuli, resulted in decreased nest-building activity and induced higher scores in the Mouse Grimace Scale. Moreover, locomotor activity and anxiety-related behaviour were strongly affected after facial vein bleeding. Interestingly, tail vessel bleeding only induced little alterations in the assessed physiological and behavioural parameters. Importantly, the observed effects in all treatment groups were no longer detectable after 24 hours, indicating only short-term impacts. Thus, by also taking the animal's perspective and comprehensively assessing the severity of the particular sampling procedures, the results of our study contribute to Refinement within the 3R concept and allow researchers to objectively select the most appropriate and welfare-friendly blood sampling technique for a given experiment.