Aim: The aim of the study was to identify sociodemographic, disease-related, physical and mental health-related determinants of fatigue at 2-year follow-up in individuals with symptomatic knee osteoarthritis (OA). Methods: A longitudinal analysis of participants with symptomatic knee OA from the Multicenter Osteoarthritis Study (MOST) was conducted to identify predictors of fatigue at 2-year follow-up. Participants self-reported fatigue at baseline for the first time in the MOST cohort and at follow-up using a 0-10 visual analog scale. At baseline, questionnaires on sociodemographics, disease-related symptoms, physical and mental health factors were completed. Data were analyzed using linear regressions with a backwards elimination approach. Results: Of the 2330 individuals in the MOST cohort at baseline, 576 had symptomatic knee OA and of these, 449 with complete fatigue values at baseline and follow-up were included in this analysis. Minimally important fatigue change (ie, worsening [≥1.13], no change [<0.82 or <1.13] and improvement [≥−0.82]) from baseline to follow-up were unequal within the population (34.5%, 26.9%, 38.5%; χ2 [2, N = 449] = 9.32, P =.009). The multiple linear regression showed that baseline fatigue (unstandardized coefficient [Β] = 0.435; 95% confidence interval [CI] 0.348-0.523, P <.001), slow gait speed (Β = −1.124; 95% CI −1.962 to −0.285, P =.009), depressive symptoms (Β = 0.049; 95% CI 0.024-0.075, P <.001) and higher numbers of comorbidities (Β = 0.242; 95% CI 0.045-0.439, P =.016) were significant predictors of greater fatigue at follow-up. Conclusion: Fatigue is strongly associated with physical- and mental-related health factors. Individualized treatments that include combined psychological and physical function rehabilitation might be modalities for fatigue management.