Negative affect (NA) and chronic stress are separately associated with health imbalances, and the interaction between the two aspects remains unclear. Care of relatives with long-term pathologies could be a likely model in the study of this issue, as caregivers frequently report chronic stress and health complaints. The aim of this study is examine the role of NA on psychophysiological stress-induced response and health in schizophrenia caregivers. Method: Forty-one caregivers were distributed into two groups accordingly to their scores in NA. Thirteen non-caregivers were included as a control group. Participants were exposed to a repeated acute psychosocial stress while salivary cortisol, immunoglobulin A (IgA), and skin conductance level (SCL) were measured before, during, and after stress. Mood, state-anxiety, care conditions and perceived general health were also assessed. Results: Caregivers with higher NA reported negative perceptions, worse health, and greater decreases in cortisol and IgA response than caregivers with low NA and than non-caregivers. Conclusions: NA could reduce the capability to develop adaptive psychophysiological stress responses. From a preventive view, the evaluation of NA could be useful to detect and assist high-risk individuals in potentially chronically stressed populations