Objective: Prospective studies have linked negative affect with hypertension, cardiovascular disease, and mortality. This study aims to identify if cardiovascular activity in day-to-day settings is related to affect levels as assessed using the Day Reconstruction Method (Kahneman, Krueger, Schkade, Schwarz, & Stone, 2004). Design: 186 people underwent baseline physiological testing and were monitored naturalistically for an entire day. Multilevel models were the principal analyses used. Main Outcome Measures: We utilized an online day reconstruction survey to produce a continuous account of affect, social interactions, and activity patterns during waking hours. Ambulatory heart rate (HR) was assessed during the same period. Personality, health behavior, consumption, self-reported activity, and baseline physiological characteristics were assessed to isolate the relationships between affect and HR. Results: Negative affect predicted an elevated ambulatory HR and tiredness predicted a lower HR. Associations between negative affectivity and increased cardiovascular reactivity were maintained after taking account of baseline physiological factors, health behavior, and personality. Conclusion: Negative affect in everyday life is a reliable predictor of HR. Combining day reconstruction with psychophysiological and environmental monitoring is a minimally invasive method with promising interdisciplinary relevance.