Oral glucose was recommended as pain therapy during venepuncture in neonates. It is unclear whether this intervention reduces excess oxygen consumption (o(2)), energy loss, or cardiovascular destabilization associated with venepuncture, and whether <2 mL glucose solution is effective. We tested the hypothesis that oral glucose solution attenuates the increases in neonatal oxygen consumption, energy expenditure (EE), and heart rate associated with venepuncture for two different volumes of glucose solution (2 and 0.4 mL). In this prospective, randomized, controlled, double-blind trial, 58 neonates (gestational age, 31-42 wk; postnatal age, 1-7 d) were randomized to 2 mL glucose 30%, 0.4 mL glucose 30%, or 2 mL water by mouth before venepuncture. The videotaped behavioral pain reactions were scored with the Premature Infant Pain Profile. Cry duration, o(2), EE (indirect calorimetry), and heart rate were measured. The 2 mL glucose solution reduced pain score and crying after venepuncture compared with controls [median pain score, 5.5 (interquartile range, 4-9) versus 11 (7-12), p = 0.01; median duration of first cry, 0 s (0-43 s) versus 13 s (2-47 s), p < 0.05, respectively]. The 0.4 mL glucose solution had no effect. The 2 mL glucose solution did not attenuate the o(2) increase during venepuncture (1.5 +/- 0.2 mL/kg min (water) versus 1.7 +/- 0.5 (0.4 mL glucose) versus 1.1 +/- 0.2 (2 mL glucose) (mean +/- SEM) nor EE nor heart rate. We conclude that oral administration of 2 mL glucose 30% before venepuncture reduced pain expression and crying, but did not prevent the rise in o(2), EE, or heart rate. Alternative therapies against the stress of nonpainful handling during venepuncture should be explored.