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To examine the association between physical fitness, cardiac parasympathetic control, and inflammatory cytokine responses to mental stress. Exercise and physical fitness may act as a buffer to the detrimental effects of psychosocial stress exposure.Participants were 207 men and women (52 ± 3 years) drawn from the Whitehall II epidemiological cohort. Participants completed two mental stressors consisting of a 5-minute Stroop task and a 5-minute mirror tracing task. Blood samples were obtained during baseline and 45 minutes post stress. Heart rate variability (HRV) was measured during baseline, stress, and recovery. Physical fitness was assessed from a submaximal exercise test.Interleukin (IL)-6 and IL-1 receptor antagonist were increased significantly at 45 minutes post stress. Multiple linear regression analysis, adjusted for age, body mass index, gender, smoking, alcohol, grade of employment, and basal levels of inflammatory markers demonstrated that exercise heart rate (a fitness indicator) was related to IL-6 (β = 0.24; p = .005) and tumor necrosis factor (TNF)-α responses to stress (β = 0.27; p = .002). Exercise heart rate was also related to the HRV response to stress (β = −0.23; p = .02). A higher systolic blood pressure response to exercise was a predictor of TNF-α responses to stress (β = 0.18; p = .03).Physical fitness (as indexed by lower exercise heart rate) is associated with smaller inflammatory cytokine responses to acute mental stress, an effect that may be partly mediated through parasympathetic pathways. This may be one of the mechanisms by which physical fitness confers protection against cardiovascular risk.