Isoflurane anesthesia alters the blood levels of several neuroendocrine hormones associated with normal metabolism and physiology and increases stress, but the effect of brief CO₂ anesthesia on these parameters is unknown. In this study, we examined the effects of isoflurane (4%) compared with brief CO₂ (70% CO₂, 30% air) anesthesia on circadian rhythms of plasma measures of physiology and metabolism. Adult male Sprague–Dawley rats (Crl:SD; n = 6 per group) were maintained on a 12:12-h light:dark (300 lx; lights on, 0600) photoperiod. After 1 wk of acclimation, a series of 6 low-volume blood draws were collected by cardiocentesis under anesthesia using isoflurane (10 min or less) compared with CO₂ (1 min or less) at a single circadian time point every 4 d (0400, 0800, 1200, 1600, 2000, or 2400) over 3 wk to assess arterial blood glucose, lactic acid, and potassium and plasma melatonin, leptin, insulin, total fatty acids, and corticosterone concentrations. Results revealed that plasma levels (mean ± SEM) of melatonin were low (11 ± 1 pg/mL) during the light phase in both groups but were significantly lower during the dark phase in the isoflurane group (48 ± 6 pg/mL) compared with the CO₂ group (162 ± 18 pg/mL). In addition, prominent circadian rhythms of arterial plasma levels of corticosterone, glucose, total fatty acids, lactic acid, and potassium were altered in the isoflurane group compared with the CO₂ group. These findings demonstrate that the normal circadian rhythms of endocrine physiology and metabolism observed during brief CO₂ anesthesia in rats are markedly disrupted by isoflurane anesthesia.