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Substrains of the C57BL/6 inbred mouse are widely used in genetic, behavioral and physiological research, as well as models for human disease. Throughout, the choice of the respective substrain can have a large influence on experimental results. Likewise, the conditions under which experiments are performed, such as the light regime, can significantly affect the outcome of an experiment, especially when aiming at experimental behavior. Here, two commonly used mouse substrains, C57BL/6JOlaHsd and C57BL/6NCrl, were housed under either a conventional or a reverse light regime and were tested in either the light phase or the dark phase, respectively. All animals were exposed to three unconditioned anxiety-related behavior set-ups: the modified Hole Board test, the light-dark box and the elevated plus maze. Significant substrain and light regime effects were found in all three behavioral tests, with some of the latter being substrain and test specific. This signifies the importance of the choice of substrain used in for example, a mouse knockout experiment studying behavior, also in relation to light regime under which the animals are tested.