Inhalation cough challenge has become an accepted method of investigating antitussive agents. It is, therefore, important to examine the degree of tachyphylaxis seen with repeated cough challenge. In addition, different types of challenge may reveal important differences in the neuronal pathways involved in the cough reflex. Citric acid, distilled water and capsaicin were examined to determine adaptation of the cough response during acute and long-term inhalation studies in healthy subjects. To study acute tachyphylaxis two separate one minute continuous inhalation challenges (n = 13) were performed. Long-term tachyphylaxis (n = 10) was examined using citric acid and capsaicin inhalation at 10 min intervals for 40 min, and at 4 and 6 h. Cross-tachyphylaxis to citric acid and capsaicin was examined in a separate randomized crossover study (n = 10). Highly significant adaptation occurred between the first and last 10 s of the one minute challenge with citric acid (90-100%) and distilled water (74-84%), but was less pronounced with capsaicin (37-49%, at 2 μM). Cough during the whole of the second test was significantly reduced for citric acid (50%). During long-term challenge cough was attenuated over 40 min both with citric acid (100 mM, p<0.05; 300 mM, p<0.001; 1 M, p<0.001) and with capsaicin (3 μM, p<0.01; 10, 30, 100 μM, p<0.001 each). With higher doses, tachyphylaxis was still present at 180 min with both citric acid (300 mM, p<0.05) and capsaicin (100 μM, p<0.008). Total cough response was diminished by 24% when citric acid was inhaled after capsaicin (p<0.05), and 33% when capsaicin followed citric acid (p<0.02). Citric acid and distilled water have a different pattern of adaptive response to capsaicin and may, thus, stimulate different receptor populations with separate afferent neuronal pathways.