Background: Multi-vessel spasm (MVS) has a prognostic impact in patients with vasospastic angina (VSA). Thus, the presence of coronary spasm in both the left coronary artery (LCA) and right coronary artery (RCA) should be assessed through the spasm provocation test (SPT). Nitroglycerin (NTG) is used to avoid SPT-related complications; however, this unavoidable use of NTG may decrease the detection of MVS. Therefore, we investigated the frequency of the unavoidable use of NTG during SPT and clarified the clinical characteristics in patients with VSA who underwent the unavoidable use of NTG during STP. Methods: A total of 141 patients with positive SPT were evaluated. A positive SPT was defined as > 90% constriction in epicardial coronary arteries in response to acetylcholine, accompanied by the usual chest symptoms and/or ischaemic ST-T changes on electrocardiography. When a coronary spasm occurred, we usually wait for the spontaneous relief of the coronary spasm. However, if a prolonged coronary spasm or unstable haemodynamics occurred, 0.3 mg NTG was administered intracoronarily to promptly relieve the coronary spasm and this was defined as the unavoidable use of NTG. Even when the unavoidable use of NTG was administered in one coronary artery, an additional SPT was performed on another coronary artery. If a coronary spasm occurred in another coronary artery, a positive SPT was diagnosed. In contrast, if a coronary spasm was not induced after the unavoidable use of NTG, the judgement was classified as undiagnosed. The patients were divided into two groups according to the unavoidable use of NTG: U-NTG (n = 42) and the final use of NTG: F-NTG (n = 99). The clinical characteristics and frequencies of MVS (≥2 major coronary arteries in which a coronary spasm was provoked) and complications (malignant arrhythmia and unstable haemodynamics requiring catecholamines) during the SPT were compared between the groups. Results: Except for smoking status, all other clinical characteristics did not differ significantly between the groups. More current smokers were observed in the U-NTG group (29%) than in the F-NTG group (12%, p = 0.02). The frequency of MVS did not vary significantly between the groups (p = 0.28), with 64% for U-NTG and 55% for F-NTG. No significant difference was found between the groups in the frequency of severe complications during SPT (p = 0.83), with 2% for U-NTG and 3% for F-NTG. In the U-NTG group, the positive induction rate of coronary spasm in another coronary artery was 40% (17/42). Conclusions: The unavoidable use of NTG occurred in ~30% of patients with VSA, most of whom were current smokers. It did not decrease the detection of MVS and potentially prevented severe complications during SPT. Therefore, the unavoidable use of NTG is acceptable during SPT. However, an additional test may need to be performed to assess the presence of MVS.