Background Hypercapnia is a threat to homeostasis and results in neuroendocrine, autonomic and anxiogenic responses. The inhalation of carbon dioxide (CO2) may, therefore, provide a good paradigm for exploring the pathways by which stress can lead to increased susceptibility to ill-health through physiological and psychological stress reactivity. The current study was designed, therefore, to assess the psychological and physiological responses to the inhalation of CO2. Methods Healthy participants (N=24) inhaled a single vital capacity breath of a mixture of CO2 (35%) and oxygen (65%). Blood pressure and heart rate were recorded for 5 min before and after the test and blood and saliva samples were taken immediately before and 2, 10, 20 and 30 min post-inhalation for the measurement of noradrenaline, salivary and serum cortisol and salivary α amylase. In addition, psychosomatic symptoms were recorded immediately before and after the test. The same protocol was repeated 4–6 weeks later at the same time of day. Results A single inhalation of CO2 increased blood pressure, noradrenaline, salivary α amylase and psychosomatic symptoms, but decreased heart rate at both testing sessions. Analyses of salivary cortisol data revealed that 70% of the sample could be reliably classified as either responders (i.e. demonstrated a post-CO2 cortisol increase) or non-responders (i.e. responded with a decrease or no change in cortisol following CO2) at both test sessions. Responders also perceived the test to be more aversive than non-responders. Conclusions Inhalation of 35% CO2 reliably stimulated the key mechanisms involved in the human stress response. The inter-individual differences in the reactivity of the hypothalamic–pituitary–adrenal axis were also related to differences in the perception of the test.