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Background: A high prestroke physical activity (PA) level is associated with reduced stroke rate, stroke mortality, better functional outcome, and possible neuroprotective abilities. The aim of the present study was to examine the possible neuroprotective effect of prestroke PA on 24-h cerebral infarct growth in a cohort of acute ischemic stroke patients treated with intravenous tPA and randomized to remote ischemic perconditioning. Methods: In this predefined subanalysis, data from a randomized clinical trial investigating the effect of remote ischemic perconditioning (RIPerC) on AIS was used. Prestroke (7 days before admission) PA was quantified using the PA Scale for the Elderly (PASE) questionnaire at baseline. Infarct growth was evaluated using MRI (acute, 24-h, and 1-month). Results: PASE scores were obtained from 102 of 153 (67%) patients with a median (interquartile range) age of 66 (58-73) years. A high prestroke PA level correlated significantly with reduced acute infarct growth (24 h) in the linear regression model (4th quartile prestroke PA level compared with the 1st quartile), β4th quartile = -0.82 (95% CI -1.54 to -0.10). However, the effect of prestroke PA was present mainly in patients randomized to RIPerC, β4th quartile = -1.14 (95% CI -2.04 to -0.25). In patients randomized to RIPerC, prestroke PA was a predictor of final infarct size (1-month infarct volume), β4th quartile = -1.78 (95% CI -3.15 to -0.41). Conclusion: In AIS patients treated with RIPerC, as add-on to intravenous thrombolysis, the level of PA the week before the stroke was associated with decreased 24-h infarct growth and final infarct size. These results are highly encouraging and stress the need for further exploration of the potentially protective effects of both PA and remote ischemic conditioning.