Rodents are the most common laboratory animals all over the world, however, most studies on the effects of laboratory conditions on the behavior and physiology of the study animals have been performed on traditional laboratory animals. We investigated the effects of environmental enrichment, lighting conditions and ambient temperature cycles on the locomotor activity of wild trapped, nocturnal Namaqua rock mice and diurnal Four-striped grass mice. When considering the general activity of the two species, the diurnal species showed more variability in locomotor activity than the nocturnal species. Cage enrichment differentially affected the intensity of the locomotor activity in the two species. Despite a decrease in activity, the diurnal species showed more structured rhythms in an enriched cage. Twilight conditions changed the behavior of both species, the active time of the nocturnal animal was contracted to the completely dark hours of the light cycle, while the active time of the diurnal species was extended in the longer daylight hours. The natural light appears to stabilize the entrainment of the diurnal species. The natural ambient temperature cycle caused changes in intensity of activity, but reinforced entrainment in both species. These results show that changes in laboratory housing conditions can affect the activity of captive wild animals and that these effects are species specific. By increasing our understanding of the effects of different environmental factors on the outcomes of experiments, both the results obtained, and the welfare of the captive animals may be improved.