Previous studies have operationalised mental effort via various indices of psychophysiology, particularly cardiovascular measures. Metabolic measures represent a complementary approach wherein mental effort investment is explicitly linked to the process of energy mobilisation. The purpose of this study was to contrast cardiovascular variables (heart rate, 0.1 Hz component of heart rate variability) with a metabolic measure (blood glucose) of mental effort. Twenty-nine participants were exposed to Stroop stimuli over a 45 min period under two conditions: (a) congruent (i.e. 100% congruent Stroop stimuli); and (b) incongruent (i.e. 100% incongruent Stroop stimuli). Performance, blood glucose, cardiovascular activity and subjective mood were measured. The results indicated that blood glucose levels were sensitive to both Stroop and time-on-task variables, whilst cardiovascular measures were only sensitive to the latter. There was also evidence of an association between blood glucose levels and response accuracy. The implications of these findings for the operationalisation of mental effort are discussed.