Preparing for a cued, speeded response induces a set of physiological changes. A review of the psychophysiology of preparation suggested that inhibition of action was an important process among the constellation of changes constituting attentive preparation. The current experiment combined event-related functional magnetic resonance imaging and cardiac inter-beat interval measures in an experiment that compared preparing for a response, watching stimuli without responding, and responding in the absence of preparation. Ten college-aged participants were tested in an initial psychophysiological experiment followed by two scanning sessions during which reverse spiral imaging was performed concurrent with inter-beat interval measurement. Two analytic approaches were used to confirm blood oxygenation level dependent responses during preparation, and these converged to show inferior prefrontal and related subthalamic nuclei activity in the context of other known changes related to brain attentional networks. Subthalamic nuclei changes were related to the depth of preparatory cardiac deceleration. This pattern of findings suggests that preparation involves the activation of a complex inhibitory neural network implicating brain and autonomic nervous systems.