BackgroundThe dose–effect relationship of Long-snake-like moxibustion for chronic fatigue syndrome (CFS) remains poorly understood. In order to address this gap, we designed this trial to assess the association between different treatment duration of Long-snake-like moxibustion and its effects on CFS based on the combination measurements of the subjective patient-reported scales with objective medical infrared imaging technology─Thermal Texture Maps (TTM).MethodsFrom December 2020 to January 2022, 60 female CFS patients were recruited and equally allocated to two groups: Group A, receiving 60-min Long-snake-like moxibustion per treatment, and Group B, receiving 30-min Long-snake-like moxibustion per treatment. The treatment was administered 3 times per week for a total of 4 weeks. The primary outcome was defined as the improvement of symptoms measured by the Fatigue scale-14 (FS-14), and secondary outcomes were designated as the improvement in Symptoms Scale of Spleen-Kidney Yang Deficiency, Self-rating depression scale, and Self-rating anxiety scale. TTM scanning was employed twice for CFS patients (before and after 4-week treatment) and once for Healthy control subjects (HCs).ResultsAt week 4, the scores of FS-14 and Symptoms Scale of Spleen-Kidney Yang Deficiency in Group A were significantly lower than those in Group B (physical fatigue: 5.00 vs. 6.00,with95%CI − 2.00 to 0.00,p= 0.003; FS-14 total score: 8.00 vs. 9.00,with95%CI − 3.00 to 0.00,p= 0.012; total score of Symptoms Scale of Spleen-Kidney Yang Deficiency: 9.80 vs. 13.07,with95%CI − 5.78 to − 0.76,P= 0.012). All thermal radiation values of the two groups increased, and statistical differences in ΔTs between Group A and HCs were not obtained. More significant correlations between symptoms improvements and ΔT changes were observed in Group A, and its ΔT changes in Upper Jiao, Shenque (CV8), Zhongwan (CV12), Danzhong (CV17), Zhiyang (GV9), Dazhui (GV14), upper arm, thoracic segments, lumbar segments, renal region, popliteal fossa strongly correlated with the improvement of Spleen-Kidney Yang Deficiency symptoms.ConclusionsIn the same course of treatment, the positive dose–effect relationship was found between the treatment duration of Long-snake-like moxibustion and CFS effect assessment. 60-min Long-snake-like moxibustion per treatment were associated with optimal clinical response and TTM improvement.Trial registrationChinese Clinical Trail Registry (No. ChiCTR2000041000, date of registration: 16 December 2020),http://www.chictr.org.cn/showproj.aspx?proj=62488Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04250-z.