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Posttraumatic stress disorder (PTSD) has been linked to elevated heart rate (HR) and reduced heart rate variability (HRV) in cross-sectional research. Recent evidence suggests that this link may be driven by individual differences in autonomic arousal associated with momentary negative affect (NA). Using ecological momentary assessment (EMA) of NA and minute-to-minute HR/HRV monitoring, we examined whether NA-related HR/HRV mediated the association of PTSD symptom severity with 24-hour HRV and endothelial functioning.One hundred ninety-seven young adults (18–39 years), 93 with PTSD, underwent 1 day of Holter monitoring while concurrently reporting NA levels via EMA. Two noninvasive measures of endothelial functioning—flow-mediated dilation and hyperemic flow—were also collected. Multilevel modeling was used to assess the associations of momentary NA with HR and low- and high-frequency HRV during the 5-minute intervals after each EMA reading. Latent variable modeling was then used to determine whether individual differences in these associations mediated the association of PTSD symptom severity with 24-hour HRV, flow-mediated dilation, and hyperemic flow.PTSD symptom severity was positively associated with NA-related autonomic arousal (β = .21, p < .001), which significantly mediated the association of PTSD symptom severity with 24-hour HRV and hyperemic flow, accounting for 62% and 34% of their associations, respectively, while overshadowing the influence of smoking, lifetime alcohol dependence, sleep duration, mean NA, and episodes of acute NA.Results suggest that NA-related autonomic arousal is both a primary factor driving cardiovascular risk in PTSD and a potential point of intervention.