Background: Despite the frequent use of the Timed Up and Go (TUG) test in clinical trials, evaluation of longitudinal test-retest reliability is generally lacking and still inconclusive for patients with Parkinson’s disease (PD). Objective: We aimed to further investigate long-term reliability and sensitivity of the TUG test among this population. Furthermore, we explored alternative assessment strategies of the test aimed at elucidating whether the inclusion or combination of timed trials may have potential implications on outcome measure. Methods: Relative and absolute reliability of the TUG performance were obtained in forty-three subjects with PD over three timed trials in two different testing sessions separated by a two-months period. Results: Our results reported excellent intra-session and moderate inter-session reliability coefficients. The use of different assessment strategies of the TUG was found to have an important impact on outcome measure, highlighting the averaging of several timed trials in each testing session as a recommended alternative to minimize measurement error and increase reliability in longitudinal assessments. Nevertheless, beyond acceptable reliability, poor trial-to-trial stability of the measure appears to exist, since the ranges of expected variability upon retesting were wide and the incidence of spurious statistical effects was not negligible, especially in longitudinal repeated testing. Conclusion: Limitations may exist in the interpretation of the TUG outputs as part of longitudinal assessments aimed at evaluating treatment effectiveness in PD population. Researchers and practitioners should be aware of these concerns to prevent possible misrepresentations of functional ability in patients for a particular intervention.