Fasting of mice is a common procedure, which can affect the outcome of the study as well as animal welfare. In this study, we assess the effects of fasting, fasting duration and fasting initiation time in relation to light schedule and present suggestions for optimization of fasting. Male C57BL/6NCrl mice were fasted for 0, 3, 6, 12, 18 and 24 hours initiated either in the light period (photophase) or the dark period (scotophase). Body weight, gastric content, body temperature, corticosterone and 19 routine clinical chemistry parameters were evaluated. Fasting caused significant changes in most of the measured parameters. Increasing duration of fasting resulted in increasing physiological changes. Fasting initiated in the scotophase caused more significant changes than fasting initiated in the photophase. To cause the least physiological changes in mice and increase animal welfare, mice should preferably be fasted in the photophase and for the shortest possible period allowed by the experimental purpose of fasting.