## 2 Materials and Methods  
The study was approved by the Research Ethics Board of the University of Chieti (reference number: 207/2015) and performed in agreement with the Declaration of Helsinki. All participants signed informed consent and could withdraw from the study at any time. Facial thermal infrared thermography (IR) of 46 volunteers (21 male and 25 female, average age 32 \(\pm\) 6.3) undergoing pressure pain threshold (PPT) of the sternocleidomastoid trigger points was recorded. Thermal IR imaging was performed using _FLIR CAM SC3000_ (NETD = 20 mk) with GaAs cooled sensor, Quantum Well Infrared Photon Detector (QWIP) 320 \(\times\) 240 pixels with 8-9 \(\upmu\)m range and thermal field range from \(-\)20 \({}^{\circ}\)C up to +80 \({}^{\circ}\)C, connected with the ThermaCAM Researcher Professional 2.9 software. The recordings were performed by observing the Academy of Neuro-Muscular Thermography's standard protocol [19]. Before performing IR imaging, participants observed acclimatisation in the measurement room for at least 20 min, the temperature of which was maintained at (23 \(\pm\) 0.5) \({}^{\circ}\)C and the humidity was 40%-50%, with no direct air ventilation or exposure to heat sources. The participants were positioned 100 cm far from the camera and were invited to sit on stiff chairs, straight, with their feet on the floor, looking straight ahead, during all the recordings. Subjects put up their hair before registration, uncovering their neck and forehead. They were asked to avoid hot/cold showers, intensive physical activity, coffee, smoking, stimulants, alcoholic drinks, and medicines able to affect sympathetic functions for at least 3 h before undergoing thermal IR imaging. Moreover, they were asked to avoid the use of cream, make-up, earrings, necklaces, and any contact between their hands and face or neck during the exam. PPT was simultaneously performed on both left and right muscles. After a 2 min baseline, an experienced operator (M.D.A) performed PPT of five TrPs locations on the sternocleidomastoid belly: (i) mastoid insertion, (ii) front cranium belly point, (iii) front caudal belly point, (iv) sternum insertion, and (v) collarbone insertion. Each PPT lasted 10 s, with an interstimulus interval of 2 min. The trigger points were stimulated in the same order across all the subjects, precisely with a cranio-caudal direction. Sixteen thermal IR imaging were recorded for each subject: at baseline (t0), 2 s before the PPT (t1), and 2 s (t2) and 60 s (t3) after the PPT of each TrPs location. During the inter-stimuli interval, avoiding any movement of head and neck, subjects were asked to draw over a head-neck template displayed on a computer screen the areas of referred pain eventually evoked by the stimulation and the referred pain intensity by means of a Visual Analogue Scale (VAS) ranked over four-levels values (0 = no pain; 1 = weak pain; 2 = clearly perceived pain; 3 = intense pain). The template was based on the innervation areas of the trigeminus nerve branches (ophthalmic, maxillary, and mandibular) and therefore divided into six areas (three for each side). The following six areas were represented in the template (Figure 1):  
- Area 1: left frontal region comprising the left nose pyramid up to the tip of it;  
- Area 2: right frontal region comprising the right nose pyramid up to the tip of it;  
- Area 3: left temporal region comprising the left hemiface cheekbone, nostril, and upper lip;  
- Area 4: right temporal region comprising the right hemiface cheekbone, nostril, and upper lip;  
- Area 5: left mandibular ramus and mandibular angle area, left hemiface, lower lip, and chin;  
- Area 6: right mandibular ramus and mandibular angle area, right hemiface, lower lip, and chin.