Background: A need exists to identify biological markers in chronic fatigue syndrome (CFS). Objective: To use an exercise and/or allergen challenge to induce the symptoms of CFS and to identify a biological marker that correlates with these symptoms. Methods: Patients with CFS (n = 32) and age-matched, normal control patients (n = 29) exercised for 20 minutes on a stationary bike at 70% of their predicted max work load (Watts). Patients from each group with positive skin test results were also challenged with intranasally administered relevant allergens. Symptoms were recorded for 2 weeks before and 1 week after each challenge, using 3 different instruments. Blood samples were taken before, and 0, 1, 6, and 24 hours after challenges. Levels of complement split products, cell-associated cytokines, and eosinophilic cationic protein were measured. Mean preexercise and postexercise symptom scores were evaluated for each group. Results: Exercise challenge induced significant increases of the complement split product C4a, but not C3a or C5a, at 6 hours after exercise only in the CFS group (P < .01), regardless of allergy status. Mean symptom scores were significantly increased after exercise through the use of a daily diary (P < .03) and a weekly diary (P < .01) for the CFS group only. Mean scores for the Multidimensional Fatigue Inventory categories “reduced activity” and “mental fatigue” were significantly increased in the CFS group only (P < .04 and P < .02, respectively). Conclusions: Exercise challenge may be a valuable tool in the development of diagnostic criteria and tests for CFS. Establishment of a role for complement activation products as markers or participants in production of illness require further study. (J Allergy Clin Immunol 2003;112:397-403.)