Materials and Methods
The protocol for this trial and supporting STROBE checklist are available as supporting information; see Checklist S1 and Protocol S1.

Ethics statements
The study was conducted in compliance with Good Clinical Practices (GCP) and the Declaration of Helsinki, and was approved by the institutional ethical review board at the University Hospital of Northern Sweden, Umeå.

Trial design
The TYNERGY study used a one-armed trial design to evaluate the natalizumab treatment effect on fatigue with a well-defined and validated instrument, the FSMC, designed for use in MS patients. A randomised controlled trial was not performed because at the time of the start of this trial there was no comparator available for the patient population with highly-active MS or with a need of second line MS therapy, which constitutes the patients fulfilling the indication for natalizumab.

Trial conduct
Consecutive patients prescribed natalizumab at the participating centers gave their written, informed consent to enter the study after the therapy decision was made. Patients were eligible for inclusion in the trial if they were prescribed natalizumab according to national guidelines, aged 18–65 years (both inclusive) at screening and presented with an FSMC sum score of ≥43 (at least mild fatigue at baseline, Table 1). Patients with no symptoms of fatigue, EDSS of ≥6, amphetamine medication or major depression, were not included.
10.1371/journal.pone.0058643.t001 Table 1  Cut-off values for the Fatigue Scale for Motor and Cognitive functions (FSMC).     The study was performed at 27 centers in Sweden (12), Norway (7), Austria (5) and Denmark (3). The patients attended 5 visits, (at baseline, month 3, 6, 9 and 12) over 12 months. The primary endpoint was fatigue associated with MS as measured by the FSMC total score change at 12 months, compared with baseline. Cut-off values for the clinical categories mild, moderate and severe MS-related fatigue are shown in Table 1. The FSMC allows for the evaluation of the physical component of fatigue, i.e. motor fatigue, as well as the cognitive component, separately, which were also evaluated at every visit and constituted secondary endpoints. To address other important aspects of the function and well-being of MS patients, secondary endpoints were assessed at baseline and at months 6 and 12. They were: Capacity for work (capacity for work questionnaire; CWQ), health related quality of life ((HRQoL) Short Form -12 questionnaire (SF-12)), sleepiness (Epworth Sleepiness Scale; ESS), depression (Center for Epidemiologic Studies Depression scale; CES-D), cognitive impairment (the Paced Auditory Serial Addition Test; PASAT, and Symbol Digit Modalities Test; SDMT), Speed of walking (6 Minute Walk Test; 6MWT), MS disease disability (Expanded Disability Status Scale; EDSS) and amount of walking e.g. a step counter was worn for seven days the week before the study visit.
The DMTs used prior to initialization of natalizumab were documented. All concomitant medications taken during the trial were recorded and special attention was paid to change in symptomatic fatigue therapy, e.g. modafinil and amantadine. Information on relapses, adverse events (AEs) and serious adverse events (SAEs) were collected.
The first patient's first visit was on March 23, 2009 and the last patient's last visit on June 30, 2011. EudraCT number for the Swedish protocol: 2008-008065-35. Clinical Trials.gov identifier: NCT00884481. The study was considered observational in Austria, Norway and Denmark.

Statistical methods
In order to achieve a power of 90% to detect 25% improvement in fatigue from baseline to month 12 when performing a paired Student's t-test, with the assumption that the standard deviation (SD) was close to 1.0, the sample size was estimated to be 168. The statistical analyses were based on pooled datasets from Sweden, Norway, Denmark and Austria, except SF-12, ESS, CES-D, PASAT and 6MWT, which were not performed in Denmark.
In general, descriptive statistics for continuous variables are presented with Mean, Standard Error (SE), Median, Minimum and Maximum values. Descriptive statistics for discrete variables are presented as percentages. All statistical tests were carried out as two-sided on a 5% level of significance unless otherwise stated.
The primary efficacy analysis was based on an analysis of variance (ANOVA) method on the change from baseline to month 12 for the FSMC total score. Baseline FSMC value was included as a continuous fixed effect.
For the secondary efficacy variables, mixed linear models with repeated measures were done for the FSMC total score, motor score, cognitive score, SF-12, ESS, CES-D, 6MWT and PASAT scores with change from baseline values as response, and baseline value as a continuous fixed effect, and visits as a categorical fixed effect. The covariance matrix was assumed unstructured. Since the EDSS step score was not normally distributed, a non-parametric model and a Hodges-Lehmann confidence interval (CI) with associated p–value from the Wilcoxon-Signed Rank test, were used to assess the change from baseline. The SDMT and Step Counter scores were analysed in a generalised linear mixed model. All statistical analysis and programming were done using SAS v9.2.