Whole body plethysmography (WBP) is used to quantify pulmonary function in conscious, unrestrained mice. We determined currently whether time of day and environmental lighting influence day-to-day reproducibility of pulmonary function, and quantifed the necessary habituation time in the WBP chamber. Two-month-old male C57BL6 and mdx mice (n = 8/group, reverse light cycle), were examined on consecutive days using a calibrated WBP chamber and manufacturer software was used to calculate respiratory measures. Respiratory data stabilized between 5–10 min for all variables. Mice exhibited time of day respiratory differences, performing more forceful and less frequent breaths midday (11:45 a.m. and 3:00 p.m.) compared to 7:30 a.m. WBP performed in darkened conditions elicited more forceful breathing than lit conditions. Day-to-day reproducibility during controlled conditions ranged from r 2 = 0.58 to 0.62 for the functional measures. Findings indicate reproducible respiratory data are obtainable following a 15-min chamber habituation and standardization of time of day and room lighting.