Background Aspirin-exacerbated respiratory disease (AERD) is diagnosed through graded aspirin challenges that induce hypersensitivity reactions and eicosanoid level changes. It is not known whether diagnostically useful changes also occur after low-dose aspirin challenges that do not induce hypersensitivity reactions. Objective To investigate the utility of low-dose oral aspirin challenges for diagnosing AERD by measuring different clinical parameters and eicosanoid changes. Methods Sixteen patients with AERD and 13 patients with aspirin-tolerant asthma underwent oral challenges with low-dose (20 or 40 mg) aspirin and diagnostic oral graded aspirin challenges (up to 325 mg of aspirin). Forced expiratory volume in 1 second, nasal peak flow, the fraction of exhaled nitric oxide (FeNO), and eicosanoid levels in plasma and urine were analyzed. Results In patients with AERD but not in those with aspirin-tolerant asthma, 40-mg aspirin challenges induced a significant mean (SEM) decrease from baseline in FeNO (19% [5.1%]; P =.001) without causing any hypersensitivity reaction. The FeNO decrease also occurred after higher-dose aspirin challenges (27.8% [4.9%]; P <.001). The sensitivity and specificity of 40-mg aspirin-induced FeNO changes for identifying AERD were 90% and 100% with an area under the curve of 0.98 (95% CI, 0.92-1.00). The low-dose challenge also induced a significant leukotriene E4 urine increase in patients with AERD (from 6.32 [0.08] to 6.91 [0.15] log-pg/mg creatinine; P <.001), but the sensitivity and specificity of these changes were less than for the FeNO changes. Conclusion The low-dose aspirin-induced decrease in FeNO in patients with AERD may be useful for the diagnosis of aspirin allergy without inducing a hypersensitivity reaction. Trial Registration clinicaltrials.gov Identifier: NCT01320072.