The traditional reinforcing-reducing manipulation of acupuncture is key for acupuncture efficacy. However, the central mechanism of reinforcing-reducing manipulations of acupuncture has not been clearly elucidated. The changes in brain activity induced by traditional reinforcing-reducing manipulations of acupuncture in healthy participants will be investigated in this study. In this study, applying multiple-channels functional near-infrared spectroscopy, a randomized crossover trial including 35 healthy participants will be conducted. The study will investigate the cerebral-response modes induced by the lifting-thrusting reinforcing manipulation, the lifting-thrusting reducing manipulation, and the even reinforcing-reducing manipulation with lifting-thrusting. The cerebral response will be analyzed with the general linear model-based activation and the seed-based functional connectivity. This study aims to partly explain the scientific connotation of acupuncture manipulations and provide a new approach to modern research on traditional acupuncture manipulations. This trial protocol was registered at the Chinese Clinical Trial Registry on October 9th, 2021 (identifier: ChiCTR2100051893).