**3. Results**

#### *3.1. Serum NfL at Time Points With and Without Active Relapse*

We categorized serum NfL levels of all time points accordingly whether an active relapse was present or not. Additionally, the change of serum NfL values at a time point with an active relapse in comparison with the previous time point was determined. Here, the absolute change of serum NfL values (pg/mL) and the percentage change was calculated (tx-tx-1). Significantly higher serum NfL levels were observed for time points with an active relapse compared with time points with no relapse (Figure 1A, *p* < 0.05). This was also true for the percentage change (Figure 1C, *p* < 0.05), but not for the absolute change of serum NfL (Figure 1B, *p* = 0.15).

**Figure 1.** Serum NfL and time points with or without an active relapse. ( **A**) Comparison of serum NfL levels during time points with and without an active relapse. (**B**) Comparison of the change of serum NfL between a time point with an active relapse and the previous time point. ( **C**) Comparison of the percentage change of serum NfL between a time point with an active relapse and the previous time point. \* *p* < 0.05.

#### *3.2. Serum NfL Levels During Follow-Up Period of 24 Months*

### 3.2.1. Patients with Relapses vs. No Relapse

In patients with a relapse-free disease course of 12 months and 24 months, serum NfL decreased significantly between baseline and time points 12 months and 18 months and time points 9, 12, 18 and 24 months, respectively (Figure 2A,B, blue triangles facing down, *p* < 0.05). There were no significant differences for baseline and follow-up visits of serum NfL levels in patients with at least one relapse within 12 or 24 months (Figure 2A,B). Furthermore, serum NfL levels in patients with a relapse within 12 months were significantly higher than in patients without a relapse within 12 months at time points 9 and 12 months (Figure 2A, *p* < 0.05).

**Figure 2.** Serum NfL levels over 12 and 24 months in patients with (red triangle facing up) and without relapse (blue triangle facing down) for (**A**) 12 or (**B**) 24 months. Symbols show median values, colored range indicates 95% confidence interval (CI), \* *p* < 0.05 for intragroup differences, # *p* < 0.05 for intergroup differences.

#### 3.2.2. Patients with EDSS Progression vs. Stable or Improved EDSS

In patients showing EDSS progression within 12 months and patients with a stable or improved EDSS for 24 months, no differences concerning their NfL levels were observed. However, in patients with a stable or improving EDSS within 12 months, serum NfL levels decreased significantly between baseline and time points 12 and 18 months (Figure 3A, *p* < 0.05). Considering 24 months of observation, patients with EDSS progression showed serum NfL levels that differed significantly from baseline serum NfL levels after 3 and 18 months (Figure 3B, *p* < 0.05).

**Figure 3.** Serum NfL levels over 12 and 24 months in patients with (red triangle up) and without EDSS progression (blue triangle down) within (**A**) 12 or (**B**) 24 months. Symbols show median values, colored range indicates 95% CI, \* *p* < 0.05.

### *3.3. Correlation of Serum NfL with*
