The primary objective of this study was to develop a cold pressor test (CPT) protocol to evaluate cardiac autonomic function. Secondary objectives were to assess CPT protocol reliability and to examine gender differences in response to orthostatic stress and the CPT. Healthy, normotensive men and women (n = 12 per group) completed 2 trials on different days in the left lateral decubitus and standing postures and during a 6 min CPT (hand immersion while seated). Measurements included R-R interval, blood pressure, ventilatory responses, spontaneous baroreflex sensitivity, and heart rate variability indices. During the CPT, blood pressure and the sympathetic nervous system (SNS) indicator increased significantly and low-frequency power, high-frequency power (normalized for tidal volume), and the parasympathetic nervous system (PNS) indicator decreased significantly. Standing caused significant increases in the SNS indicator and decreases in the R-R interval in both genders. The PNS indicator was higher in women than in men in the left lateral decubitus posture. The 6 min hand-immersion CPT provoked cardiac sympathetic activation and parasympathetic withdrawal; however, it is best suited to studies with a repeated measures design, as analysis of reliability suggests that responses are highly variable between individuals. Performing the CPT in the left lateral decubitus position may prevent vasovagal responses.