The relationships between positive affect, salivary cortisol over the day, and cardiovascular responses to laboratory mental stress tests, were assessed in 72 healthy non-smoking men (mean age 33.6+/-8.8 years). Positive affect was measured by aggregating ecological momentary assessments (EMA) of happiness obtained at four times on each of 2 working days, and by questionnaire using the Positive and Negative Affect Schedule (PANAS). Saliva was sampled on 2 days, on waking, 30 and 60 min later, and four other times over the day. Blood pressure and heart rate responses to speech and mirror tracing tasks were measured over two sessions 4 weeks apart. Data were analysed using regression of positive affect on biology adjusting for age, body mass and negative affect, with additional adjustment for time of waking in cortisol analyses and for work stress in cardiovascular analyses. EMA positive affect was inversely associated with cortisol early in the day and with the cortisol increase after waking, controlling for age, body mass index, and negative affect (P=0.012). There was no relationship between PANAS positive affect and cortisol, or between EMA positive affect and cortisol later in the day. Diastolic pressure recovery post-stress was more rapid among participants with high positive affect (P=0.022) and with lower systolic pressure throughout the stress sessions, after controlling for covariates including negative affect. PANAS positive affect was also inversely associated with systolic pressure, but not with diastolic stress or heart rate. We conclude that positive affect is related to biological responses in the laboratory and everyday life that may be health protective. Effects were substantially stronger when positive affect was assessed by aggregating EMA samples than with questionnaire measures.