INTRODUCTION: Post-stroke fatigue (PSF) is common and impacts stroke rehabilitation. Dopaminergic treatment may have beneficial effects on PSF. This study investigated whether levodopa, compared with placebo, was associated with a lower frequency or severity of PSF during in-hospital rehabilitation. PATIENTS AND METHODS: Enhancement of Stroke Rehabilitation with Levodopa (ESTREL)-Fatigue was an exploratory analysis of secondary outcome data obtained in the multicentre, randomised, placebo-controlled ESTREL trial. Participants with acute stroke received levodopa 100 mg/carbidopa 25 mg or placebo 3 times daily for 39 days to enhance motor recovery. Participants who (i) reported fatigue at 5 weeks and who (ii) took at least 80% of the study medication were included in ESTREL-Fatigue. No adjustments for confounding were made. The primary endpoint was the presence of PSF at 5 weeks, defined as a T-score of ≥ 55 on the Patient-Reported Outcomes Measurement Information System (PROMIS) Fatigue-Short-form-4a. As secondary endpoints, T-score cutoffs of ≥ 60 (moderate fatigue) and ≥ 70 (severe fatigue) were used. Binary logistic regression was used to compare PSF at 5 weeks between treatment groups. Results are presented as odds ratios (ORs) with 95% CI. RESULTS: A total of 456 of 505 (90.3%) participants were included (levodopa/placebo 235/221, median age 73 years, 41% female). Post-stroke fatigue at 5 weeks was present in 63/235 (26.8%) levodopa-treated participants and in 65/221 (29.4%) placebo-treated participants (OR: 0.88; 95% CI, 0.58-1.32; risk ratio 0.91; risk difference - 2.6%). For cutoffs of ≥ 60 and ≥ 70, ORs were 0.78 (95% CI, 0.43-1.41) and 0.8 (95% CI, 0.25-2.44), respectively. A sensitivity analysis as per intention-to-treat with all 610 randomised ESTREL participants also showed no significant difference in fatigue presence between levodopa and placebo groups (OR: 0.95; 95% CI, 0.65-1.39) and a sensitivity analysis using a mixed-effects logistic regression showed no evidence of centre-related clustering. CONCLUSION: In ESTREL-Fatigue, levodopa, compared to placebo, was not associated with less PSF during in-hospital rehabilitation.