Background: Accelerometers and patient-reported outcomes (PRO) are used to assess physical activity (PA) in people with multiple sclerosis (pwMS). So far it is unknown, however, whether these assessments represent mobility limitations in pwMS with mild and moderate to severe disability alike. The primary aim of the study was to assess the correlation between accelerometry and International Physical Activity Questionnaire (IPAQ) scores in pwMS with different degrees of ambulatory impairment. Taken its frequent use into account, the Godin Leisure Time Exercise Questionnaire (GLTEQ) was investigated as additional PRO. Methods: In a prospective cohort of pwMS, correlational analyses were performed between the number of daily steps, time spent in light, moderate to vigorous PA (MVPA) and time spent sitting as assessed using accelerometry (ActiGraph (R)-GT3X), and the respective IPAQ and GLTEQ scores. Additionally, associations of PA with disease-specific characteristics, aerobic capacity (VO2(peak)), walking assessments (Timed 25-Foot Walk, T25FW; 2-Minute Walk Test, 2MWT) and walking perception (Multiple Sclerosis Walking Scale-12; MSWS-12) were explored. Patient subgroups with mild (Expanded Disability Status Scale; EDSS score <4.0) and moderate to severe disability (EDSS >= 4.0) were analysed for the impact of ambulatory impairment on PA. Multiple linear regression was used to determine predictors of PA. Results: A total of 56 pwMS completed the study, with a mean (standard deviation, SD) age of 48.4 (10.3) years, disease duration of 14.8 (9.6) years and median (interquartile range) EDSS score of 3.5 (2.0 - 4.4). Moderate to weak correlations were found between daily step count and IPAQ total metabolic equivalent (MET) minutes/week (p < 0.001; r = 0.506), MVPA MET-minutes/week (p < 0.01; r = 0.479) and walking MET-minutes/week (p < 0.05; r = 0.372) in the total cohort. Time spent sitting was inversely correlated with total MET-minutes/week and MVPA MET-minutes/week (p < 0.05; r = -0.358 and r = -0.365). Subgroup analysis revealed, that daily step count was significantly correlated with total MET-minutes/week, MVPA MET-minutes/week and walking MET-minutes/week (p < 0.01, r = 0.569; p < 0.01, r = 0.531 and p < 0.05, r = 0.480, respectively) in the "mild disability" subgroup only, whereas time spent sitting was inversely correlated with total MET-minutes/week (p < 0.05; r = -0.582) in the "moderate to severe disability" subgroup. There was no association between objectively assessed PA and GLTEQ scores in any group. In the total cohort, moderate to weak correlations were found between daily step count and walking assessments (T25FW: p < 0.01, rho = -0.508; 2MWT: p < 0.01, rho=0.463) and MSWS-12 (p < 0.001; rho = -0.609). Moderate to weak correlations were also observed between VO2(peak) and walking assessments (T25FW: p < 0.01; rho = -0.516; 2MWT: p < 0.01, rho=0.480). Multiple linear regression analysis identified disability and VO2(peak) as predictors of PA (p = 0.045; beta=0.25 and p < 0.001; beta=0.49). Conclusion: Significant associations of objective PA measurements using accelerometry with IPAQ were found only in pwMS with "mild disability". In pwMS with "moderate to severe disability", IPAQ did not reflect the objectively assessed amount of PA. In our cohort, GLTEQ showed no association with objectively assessed PA. Thus, an MS-specific self-reported questionnaire for assessing PA is warranted.