BACKGROUND: Heart rate variability (HRV) measures homeostatic regulation of the autonomic nervous system in response to perturbation and has been previously shown to quantify risk for cardiac events. Despite known interactions among stress vulnerability, psychiatric illness, and cardiac health, however, this is the first study to our knowledge to compare directly the value of laboratory HRV in predicting autonomic modulation of real-world emotional stress. METHODS: We recorded electrocardiograms (ECG) on 56 subjects: first, within the laboratory and then during an acute emotional stressor: a first-time skydive. Laboratory sessions included two 5-min ECG recordings separated by one ambulatory 24-hour recording. To test the efficacy of introducing a mild emotional challenge, during each of the 5-min laboratory recordings, subjects viewed either aversive or benign images. Following the laboratory session, subjects participated in the acute stressor wearing a Holter ECG. Artifact-free ECGs (n = 33) were analyzed for HRV then statistically compared across laboratory and acute stress sessions. RESULTS: There were robust correlations (r = .7-.8) between the laboratory and acute stress HRV, indicating that the two most useful paradigms (long-term wake, followed by short-term challenge) were also most sensitive to distinct components of the acute stressor: the former correlated with the fine-tuned regulatory modulation occurring immediately prior and following the acute stressor, whereas the latter correlated with gross amplitude and recovery. CONCLUSIONS: Our results confirmed the efficacy of laboratory-acquired HRV in predicting autonomic response to acute emotional stress and suggest that ambulatory and challenge protocols enhance predictive value.