© 2019 Elsevier B.V. Recent research has suggested that diminished, as well as elevated reactivity to acute psychological stress is maladaptive. These differences in stress reactions have been hypothesized to relate to the Big Five personality traits, which are said to be biologically-based and stable across adulthood; however, findings have been inconclusive. This study sought to replicate the findings of the largest study conducted to date (Bibbey et al., 2013), with a sample of participants from the Midlife in the United States Study (MIDUS), aged between 35 and 84 years (M = 56.33, SD = 10.87). Participants (N = 817) undertook a standardized, laboratory-based procedure during which their cardiovascular and neuroendocrine reactivity to acute stress was measured. In contrast to Bibbey et al. (2013), associations between neuroticism and blunted reactivity did not withstand adjustment for confounding variables. Further, following adjustment for multiple tests, no significant positive association between agreeableness and HR reactivity was observed. Methodological differences between the studies, which may account in part for the contrasting findings, are discussed. Further conceptual replication research is needed to clarify associations between the Big Five personality traits and stress reactivity, across the lifespan.