We investigated the temperamental traits high-intensity pleasure (temperamental activation) and shyness (temperamental inhibition) in relation to autonomic function as measured by heart rate (HR), respiratory sinus arrhythmia (RSA), and baroreflex sensitivity (BRS) in 938 10-13-year-old preadolescents from a population cohort. Temperament was evaluated by parent reports on the Revised Early Adolescent Temperament Questionnaire. Autonomic measurements were obtained in supine and standing position. High-intensity pleasure was negatively associated with supine HR and positively with supine RSA and BRS in both genders. Shyness was positively related to supine BRS in girls only. Orthostatic-based autonomic reactivity (difference) scores adjusted for supine values were unrelated to temperamental measures. It appeared that higher scores on temperamental activation and inhibition are associated with higher cardiac vagal activity (RSA) and/or flexible regulation of autonomic balance (BRS), implicating healthy physiological functioning. Moreover, results suggest a physiological basis promoting the tendency towards engagement in high-intensity activities.