Burrowing and nest building are examples of highly motivated innate behaviours in rodents. Assessments based on these behaviours can be used as non-invasive indicators of pain, distress and suffering. In this study, we investigated the effect of three stressful procedures, namely grid floor housing for 24 h, 15 min of isoflurane anaesthesia and an intraperitoneal saline injection daily for three days, on nest building and burrowing in female C57BL/6NTac mice. For burrowing, we also investigated the effect of the presence of a shelter and nesting material (standard home cage enrichment) versus no shelter and nesting material, and whether the test can be performed during normal working hours. Alongside this, we investigated the effect of grid floor, isoflurane anaesthesia and intraperitoneal injections on stress hormone level, body weight, fur status and sucrose preference. The burrowing test was found to be sensitive to 24 h grid floor housing when no shelter but only a cardboard tube was present during testing. The mice burrowed a mean of 21 g less after grid floor housing (P <.01). This change was accompanied by decreased sucrose preference (P <.01) and body weight (P <.01). 15 min of anaesthesia with isoflurane induced changes in the nest building activity test. After exposure to isoflurane the mice built less complex nests (P =.04). This was accompanied by a decrease in sucrose preference (P <.01), a decrease in body weight (P <.01), and elevated stress hormone levels (P <.01). One daily intraperitoneal injection of saline for three days did not result in changes in nest building activity (P >.01). We also found that the mice burrowed equal amount during normal working hours and prior to the dark phase, indicating that the burrowing test can be performed during normal working hours instead of prior to the dark phase (P =.62).