Chronic venous cannulation in mice is an acceptable and useful technique for repeated blood sampling or continuous intravenous administration of substances, for which mild restraint of the animal may be necessary. Because chronic restraint has drawn considerable attention in the animal welfare community, the purpose of this study was to evaluate physiologic indices of stress in mice restrained by using an established tail restraint method. Serum corticosterone levels and body, thymus, adrenal, and spleen weights on days 2, 5, 8, and 12 were compared between tail-restrained and unrestrained mice. There were no significant differences between the two groups at the time points evaluated. Corticosterone levels were highest on day 8 for both groups and were significantly different from those on days 2 (P<0.009) and 5 (P<0.04) for restrained mice and on day 2 (P<0.02) for unrestrained mice. Levels in both groups declined by day 12, suggesting habitation. Weight loss was observed in all mice whether restrained or unrestrained. Significant differences in body, thymus, adrenal, and spleen weights were not evident between restrained and unrestrained mice. This study provides important information for balancing issues of prolonged restraint, animal well-being, and research goals.