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Background and purpose: As a high proportion of people with clinically isolated syndrome (pwCIS) exhibit sympathetic adrenergic and sudomotor dysfunction, the aim of this study was to investigate the evolution of autonomic nervous system (ANS) abnormalities in pwCIS over a 2‐year follow‐up. Methods: This was a prospective cohort study in which 121 pwCIS were enrolled and followed for 2 years. After 2‐year follow‐up, data were available for 84 pwCIS. ANS symptoms were evaluated with the Composite Autonomic System Score‐31 (COMPASS‐31) and results of the ANS tests were expressed using the Composite Autonomic Scoring Scale (CASS) at baseline and visit at month 24. Symptomatic dysautonomia was defined if the patient had a COMPASS‐31 value above the median of the whole cohort at baseline evaluation (COMPASS‐31 > 6.79) and CASS score >0. Results: Complete CASS data at baseline and month 24 were available for 62 patients; in 24 (38.7%) patients there was worsening, in 16 (25.8%) there was improvement and in 22 (35.5%) there was no change in CASS score. In 90% of pwCIS (72 of 80) there was no change in parasympathetic nervous system tests, whereas 47.3% (35 of 74) had either worsening or improvement in sympathetic adrenergic and 28.6% (20 of 70) had either worsening or improvement in sudomotor function. A multivariable regression model identified the total number of T2 lesions as an independent predictor for worsening of symptomatic dysautonomia. No predictors for worsening or improving of CASS score were identified. Conclusion: A substantial proportion of pwCIS experienced worsening of ANS abnormalities during the 2‐year follow‐up and magnetic resonance imaging parameters seemed to predict these abnormalities. [ABSTRACT FROM AUTHOR]