## 3 Results  
### Statistical and Exploratory Analyses  
A Shapiro-Wilk normality test showed that weight (MS: \(p\) = 0.251, control: \(p\) = 0.220), height (MS: \(p\) = 0.366, control: \(p\) = 0.463, and BMI (MS: \(p\) = 0.234, control: \(p\) = 0.414) were normally distributed and did not differ between groups. Age was not normally distributed (MS: \(p\) = 0.017, control: \(p\) = 0.077), but was similar between groups (Mann-Whitney U Test U = 89.0, \(p\) = 0.837). A Chi-Square test showed that sex had a similar distribution between groups (\(\chi^{2}\) = 1.463, \(p\) = 0.227), thereby confirming the validity of the sex- and age-matching procedure.  
The spectral data were collected from both extracranial and cranial sites without causing any discomfort to the participants. A total of 140 spectral acquisitions were obtained from every subject, consisting of 100 recordings from extracranial sites and 40 from cranial sites. This resulted in a comprehensive dataset comprising 3640 spectra, with 2600 spectra obtained from extracranial sites and 1040 from cranial sites.  
Figure 2 shows the mean reflectance spectra recorded from the analyzed sites, while pre-processed spectra are reported in Supplementary Materials (Figure S1). A comparison between the mean and standard deviation of the raw reflectance and pre-processed (EMSC + GLS-W + MC) spectra for each analyzed anatomical site is reported in Figure S2 (Supplementary Materials). A visual inspection of the average spectra revealed differences between groups and measurement locations. For cranial sites, at the cortical site the most evident spectral features are in the range 1050-1100 nm, around 1500 nm, and in the ranges 1900-2000 nm and 2200-2400 nm; at the brainstem site, in the range 1100-1300 nm, around 1800 nm, and between 2200 and 2300 nm. For the extracranial sites corresponding to the biceps and triceps, the most prominent spectral variations were observed at specific wavelengths, particularly around 1050 nm, within the 1300-1400 nm range, in the range 1600-1800 nm, around 1900 nm, and around 2200-2300 nm.  
Figure 2: Mean reflectance spectra acquired from extra-cranial and cranial anatomical locations: (**a**) MS biceps vs. Normal biceps, (**b**) MS triceps vs. Normal triceps, (**c**) MS cortical vs. Normal cortical, and (**d**) MS brainstem vs. Normal brainstem.  
PCA performed on the dataset associated with the examined site pairs demonstrated that spectral scores were grouped according to specific class pairs: \"MS cortical/Normal cortical\", \"MS brainstem/Normal brainstem\", \"MS biceps/Normal biceps\", and \"MS triceps/Normal triceps\". Across all cases, PC1 accounted for the highest variance among the analyzed classes. The PCA score plots indicated that the explained variance (EV) for PC1 varied across the different anatomical site pairs, with values of 38.85% for the biceps (Figure 3a), 47.96% for the triceps (Figure 4a), 25.93% for the cortical site (Figure 5a), and 33.33% for the brainstem site (Figure 6a).  
Figure 4: Principal Component Analysis (PCA) score plots of the first two principal components (**a**) derived from spectra collected at the extra-cranial triceps site in both patients (MS) and healthy subjects (Normal). Panel (**b**) presents the loadings plot for the first principal component.  
Figure 3: Principal Component Analysis (PCA) score plots of the first two principal components derived from spectra obtained at the extra-cranial biceps site in both multiple sclerosis (MS) patients and healthy controls (Normal) are presented in (**a**). The corresponding loading plot for the first principal component (PC1) is shown in (**b**).