AbstractPurposeTo examine whether the interaction between exercise intensity and sleep duration/quality is associated with heart rate variability (HRV).MethodsA sample of 391 adults (Mage = 57 years) from the Midlife in the United States Biomarker study 2004–2009 provided sleep actigraphy, electrocardiogram (ECG) HRV measurements, completed the Pittsburgh Sleep Quality Index (PSQI), and answered questions on exercise habits. Participants were grouped as short (< 6 h) or non-short sleepers (≥ 6 h), poor (> 5 PSQI global score), or good sleepers (< 6 PSQI global score), and exercise was divided into vigorous (VPA) and moderate (MPA) intensities. Based on CDC guidelines, VPA was classified into adequate (≥ 75 min/week) and inadequate (< 75 min/week) groups. For MPA, adequate (≥ 150 min/week) and inadequate (< 150 min/week) groups. Linear models, adjusted for sociodemographic and health-related covariates, examined the interaction between sleep duration/quality and exercise on HRV.ResultsInadequate VPA was associated with lower HRV, HF-HRV (B = − 0.25, SE = 0.09, p = 0.007), and RMSSD (B = − 0.15, SE = 0.05, p = 0.0009). MPA showed no significant main associations with HRV. Sleep duration/quality did not show direct associations with HRV; however, interactions were found with sleep duration. Among short sleepers, inadequate VPA was associated with lower HF-HRV (B = − 0.62, SE = 0.25, p = 0.01) and inadequate MPA was associated with lower RMSSD (B = − 0.26, SE = 0.10, p = 0.01) compared to adequate exercise. Among non-short sleepers, there were no significant differences in HRV between exercise groups.ConclusionThese findings suggest that short sleep and inadequate exercise may interact to lower HRV.