This study was conducted in two sessions spaced 24 hours apart. All measurements were conducted in the same university laboratory. Skin temperature variations following a suprathreshold pressure stimulation (STPS) at the infraspinatus muscle were assessed using an infrared thermography camera at baseline (Day 0) and 24 hours after (Day 1) where the participants had exercise-induced muscle pain (Fig. 1).

The protocol adhered to the guidelines established by the American Academy of Thermology [25]. The two sessions were identical except at the end of Day 0, where participants performed an eccentric exercise for the external rotators of the shoulder with the aim of generating exercise-induced muscle pain the following day. This has been demonstrated to be a good experimental pain model for mimicking clinical pain and provoking temporary pain sensitization [5,9,26].

The study complied with the Declarations of the Helsinki World Medical Association and reported following the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) statement for observational studies. All participants signed written informed consent. The local Ethics Committee for Clinical Research approved the study (Act No. CP15/2015) which was registered at ClinicalTrials.gov (NCT04097249).

The study sample consisted of healthy participants of both sexes recruited through advertisements in a university population and on social media. Participants were included if they were in the age range of 18 to 65 years and reported being pain-free (in the shoulder and/or in general), without a history of pain in the last six months, or previous history of fracture or surgery in the upper limbs or neck. Individuals with any systemic or regional pathology (e.g., spinal stenosis, nerve lesions, Raynaud’s syndrome) that might interfere with the evaluation or presented a psychological disorder, pregnancy, or were under pharmacological treatment were not included in the study. In addition, participants were excluded if they presented wounds, tattoos, or scars over the upper limb. Participants were instructed not to use nasal decongestants, analgesics, or anti-inflammatory medication prior to the experimental sessions. Likewise, participants were instructed not to use any substances that might affect the function of the sympathetic nervous system such as caffeine, alcohol, or smoking two hours before the assessment in each session.

At the beginning of each session, the pressure pain threshold (PPT) at the infraspinatus muscle on the right side was determined using a digital algometer (Somedic SenseLab AB, Hörby, Sweden) with a flat 1 cm2 probe area (covered with a latex sheath). Determining PPT has proven to be a highly reliable method to test pain sensitivity [27] and, as such, can be used to determine the amount of mechanical stimulus needed to evoke pain. PPT measurements were conducted in sitting, with the contralateral (left) forearm supported and elbow flexed at 90º. The assessed limb was in a neutral position, avoiding contact against any surface (as this could bias thermographic measurements). The PPT was assessed at the midpoint of the infraspinatus muscle belly using the midpoint of the spine of the scapula as a reference [5,9]. The point was marked to ensure that the following measurements were made at the same site. The infraspinatus muscle was selected, as previous studies have demonstrated a characteristic extensive referred pain following a nociceptive stimulus to the muscle of the shoulder, upper arm and forearm [5,24,28]. To determine the PPT, the pressure was gradually increased at a rate of 30 kPa/s, where the participant was instructed to press a button at the first instance the pressure became slightly painful. Three measurements were performed with a 30-second interval between measurements, and the mean value was calculated and extracted for data analysis.

The mean PPT value was used to calculate the amount of force needed to apply during the STPS, which was done at 20% over the PPT and was used to evoke referred pain from the infraspinatus muscle. The STPS was performed with the algometer and sustained for 60 seconds in accordance with previous studies [5,9]. Immediately after STPS, the participants were asked to indicate pain distribution by shading the area of experimental pain on a printed body chart. For data analysis, the upper limb was divided into three regions: 1) the shoulder region comprising the area between the stimulation site and the insertion of the deltoid muscle on the deltoid tuberosity; 2) the arm region, from the insertion of the deltoid muscle to the axis of the elbow joint; and 3) the forearm region, from the axis of the elbow joint to the fingertips (Fig. 2a). In line with previous studies, referred pain was considered to be any pain occurring outside the stimulation area [5].

To assess sympathetic vasomotor activity in relation to STPS, an infrared thermography camera (FLIR Thermacam E60; FLIR Systems, Boston, MA) with a specific software (FLIR Tools-Software; FLIR Systems) was used to assess the temperature variations in the area of the experimental referred pain. The room temperature ranged from 24–25°C and the humidity was between 45% and 50% in both sessions. Before starting data collection, the participants stood quietly for 15 minutes in the room to obtain a stable body temperature and to ensure correct camera set-up [25] before initiating the assessments. All thermography recordings were performed by a person with extensive experience in using the technique (CVC). Thermography was performed by taking two snapshots in each session, before (pre-STPS) and immediately after the STPS stimulation ceased (post-STPS). On Day 0, a thermographic picture was taken of both upper limbs to confirm that no side differences were present. All pictures were taken in a standing position at a one-meter distance from the participant. All participants were assessed with the shoulder girdle and the upper arm exposed and were instructed not to touch their upper limbs throughout the entire duration of the assessment.

For analysis of thermographic images, the same division of the upper limb was used as for the distribution of referred pain: shoulder region, arm region, and forearm regions (Fig. 2b). The mean temperature of each area of interest was extracted, taking into account that 0.3°C was the margin of error for the thermography equipment [20,22].

At the end of Day 0, all participants performed an eccentric exercise of the external rotators of the shoulder to provoke exercise-induced muscle pain in a similar manner as described previously [5,9], by performing 4 sets of 10 repetitions of eccentric contractions against the resistance of an elastic band. The participants stood in a stable position with a straight back and the elbow flexed at 90°. Twenty-four hours later (Day 1), the participants completed a 7-item Likert scale to evaluate the level of exercise-induced muscle pain (0 = no soreness; 1 = dull feeling of soreness; 2 = light, continuous soreness; 3 = more than light soreness; 4 = annoying soreness; 5 = severe soreness; 6 = intolerable soreness). This scale has been frequently used in studies to assess DOMS [29].

A sample size calculation was conducted using G*Power (v3.1.9.2; Heinrich-Heine-University, Dusseldorf, Germany) with a significance level of 0.05, a power of 90% and a desired medium effect size (Cohen’s f of 0.25) to detect a difference of 0.3°C [22,23]. As the correlation between repeated measures of infrared thermography image analysis is not lower than 0.75 with a conservative non-sphericity correction of 0.7 [30], a minimum of 20 participants were required.

The statistical analysis was performed with SPSS Statistics (ver.25; IBM Co., Armonk, NY) by a person blinded to the study hypothesis and collected data (PBL). Data are presented as mean and standard deviation (SD) or median and interquartile range (IQR) depending on the distribution of the data which was evaluated using the Shapiro–Wilk tests. Greenhouse–Geisser correction was applied when the assumption of sphericity was violated. The significance level for all statistical tests was set at P ≤ 0.05.

A paired sample t-test was used to ensure that no side differences in temperature were present at baseline. A paired sample t-test was used to analyze changes in PPT at the infraspinatus muscle between Day 0 and Day 1. Chi-square tests were used to assess differences in the frequencies of the regions affected by referred pain after STPS between Day 0 and Day 1. The Wilcoxon signed-rank test was used to compare the total number of regions affected by referred pain on Day 0 and Day 1. The Mann–Whitney U-test (MWU) was used to compare the total number of regions affected by referred pain between males and females at Day 0 and Day 1. Furthermore, the MWU was also used to compare the Likert scale scores for exercise-induced pain (DOMS intensity).

A repeated-measures analysis of covariance (RM-ANCOVA) was used to investigate temperature values with time (pre-STPS and post-STPS), site (shoulder, arm, and forearm regions), and day (Day 0, Day 1) as within factors. A RM-ANCOVA was used to evaluate variations in temperature before and after STPS (“temperature value post-STPS” minus “temperature value pre-STPS”) with site and day as repeated factors. Besides, the effect of sex was analyzed in both previous models by introducing sex as a covariate. Additionally, a mixed model analysis of variance (ANOVA) was used to explore specific sex interaction in temperature variations and PPTs with sex (male and female) set as a fixed factor and site and day as repeated factors. Bonferroni correction was used as a post hoc test to account for multiple comparisons.

Spearman’s rank correlation coefficient was calculated to display the relationship between binomial temperature variations according the margin of error of the thermography equipment (“No”: a variation lower than 0.3°C; “Yes”: a variation equal to or higher than 0.3°C) and the number of body regions affected by referred pain following STPS.

A significant interaction between site, time, and day was detected (RM-ANCOVA, F_(2,38) = 3.35, P = 0.046) in absolute temperature values, whilst no effect of sex was detected (P = 0.350). On Day 0, the values of temperature pre-STPS were higher than temperature post-STPS on the arm (Bonferroni: P = 0.001; CI, 0.13 to 0.45) and forearm (Bonferroni: P = 0.003; CI, 0.11 to 0.50). On Day 1, pre-STPS temperature was higher than post-STPS temperature on the shoulder (Bonferroni: P = 0.015; CI, 0.05 to 0.37), arm (Bonferroni: P = 0.001; CI, 0.15 to 0.50) and forearm (Bonferroni: P = 0.010; CI, 0.07 to 0.46). Temperature values on the shoulder region were higher than temperature values on the arm (Bonferroni: P < 0.001; CI, 0.29 to 0.76) and forearm (Bonferroni: P = 0.010; CI, 0.13 to 0.74) on both days (Table 1).

For temperature variations, a significant interaction was detected between site and day (RM-ANCOVA, F_(2,38) = 3.35, P = 0.046). On Day 0, the decrease in temperature at the shoulder was lower compared to the arm (Bonferroni: P = 0.001; CI, 0.11 to 0.37) and forearm (Bonferroni: P = 0.018; CI, 0.04 to 0.46). On Day 1, no significant differences were found in the decrease in temperature among the body regions (Fig. 3). Additionally, a sex interaction was detected (P = 0.010), suggesting that male and female present different variations in temperature.

Fig. 4 shows variations in temperature before and after STPS on Day 0 and Day 1, divided by sex. The mixed model ANOVA revealed a significant interaction between site and sex (ANOVA, F(2,36) = 3.44, P = 0.043). On Day 0, temperature decrease after STPS in females was greater than in males in the forearm (Bonferroni: P = 0.039; CI, 0.02 to 0.72). On Day 1, a greater temperature decrease was found amongst females compared with males at the shoulder (Bonferroni: P = 0.018; CI, 0.07 to 0.63), arm (Bonferroni: P = 0.046; CI, 0.01 to 0.69) and forearm (Bonferroni: P = 0.005; CI, 0.17 to 0.81). A greater decrease in temperature was likewise found in females at the shoulder on Day 1 compared to Day 0 (Bonferroni: P = 0.050; CI, 0.00 to 0.58).

Following STPS on Day 0, 55% of participants experienced referred pain in the shoulder region, 35% in the arm region, and 25% in the forearm region. On Day 1, 70% of participants experienced referred pain in the shoulder region, 55% in the arm region, and the 20% in the forearm region. The frequencies of referred pain in each region did not differ between days (Fig. 5). However, the total number of body regions affected by pain on Day 1 (1; 1–2 IQR) was larger compared to Day 0 (1; 1–1 IQR) (T = 21.0; z = –2.45; P = 0.014) in the total group. The number of regions affected by experimental referred pain was higher in females than in males on Day 0 (MWU = 28.0; z = –2.19; P = 0.029), while there were no differences between the sexes on Day 1 (MWU = 49.0; z = –0.90; P = 0.929). A negative correlation between the number of body regions affected by referred pain and binomial temperature changes after STPS was found in the arm and forearm regions on Day 1, revealing that participants who displayed a temperature reduction greater than 0.3°C were more likely to experience a higher number of referred pain areas. No significant correlation was found on Day 0 (Table 2).

The participants in this study demonstrated a decrease in skin temperature following STPS of the infraspinatus muscle (Fig. 2, Table 1). In a previous study investigating referred pain and thermography, Kruse and Christiansen [31] observed that a 60 sec pressure stimulation at tolerance level caused a significant temperature decrease in the areas of referred pain across the entire upper limb. The greater temperature decrease and higher number of limb regions with temperature change, found by Kruse and Christiansen [31], may relate to differences in stimulation intensity where this study only applied a stimulus 20% above PPT. Later studies using similar methods have shown both a skin temperature decrease in the referral area [32] or no change [12], and that painful stimuli could affect sympathetic vasoconstrictive activity and prevent primary neural vasodilation. In line with our results, Srbely et al. [15] found a temporal relationship between experimentally induced central sensitization and skin temperature decrease within segmentally linked dermatomes in healthy individuals. Furthermore, the present study induced referred pain in both basal and sensitized (DOMS) conditions, which is an important novelty compared to previous results since pain sensitization has been linked to larger areas of experimentally induced referred pain [5,26]. The authors believe that their findings support central sensitization possibly being a contributing mechanism in the clinical expression of sympathetic responses in humans. Overall, these findings can be interpreted as strong nociceptive stimuli causing a global change in vasomotor activity [18], which may relate to a general response to threat. The present study, however, demonstrated a unilateral change in cutaneous blood flow, although the relatively low stimulation intensity used here might have been so low that the entire variations are not significant. However, it is worth noting that the intensity of the muscle stimulation as a factor partially explaining these diverse results is a hypothesis grounded in previous studies showing larger pain durations following continuous versus bolus injections of hypertonic saline [4].

In contrast, recent studies have shown that a vasodilation occurs in the area of referred pain when applying a nociceptive stimulus to sore muscle [33,34] and that increases in overlying skin blood flow occur at the stimulation site, but not in distant regions in healthy individuals [35,36] and fibromyalgia patients [37]. From there, it was hypothesized that the variations in cutaneous blood flow might be related to the increased pain sensitivity and increased sympathetic activity [37,38]. To potentially better understand this discrepancy, it is worth noting the dynamic of a normal vasomotor response following noxious stimulation of a healthy muscle. Here, an immediate decrease in skin blood flow is seen, which normalizes approximately 15 minutes after the stimulation and is followed by an increase in such skin blood flow [18,39].

However, despite the present study showing temperature decreases after STPS at baseline and in sore conditions in the area of the referred pain, the temperature variations at baseline and in male participants at all timepoints were below the margin of error of the thermography camera, while the variations in the sore condition were above that margin. Furthermore, the negative correlation between the temperature variations and the number of regions affected by experimental referred pain was found only in the exercise-induced muscle pain condition.

In summary, the current and previous findings indicate that the degree of sympathetic vasomotor responses are related to the stimulation intensity, where more intense stimuli result in the greatest temperature changes [18]. Importantly, the level of pain sensitization due to peripheral and central mechanisms produced by DOMS [5,40] may further amplify the response; not only resulting in expanded referred pain [5] but also in greater vasomotor responses.

At baseline, and between days, females displayed greater temperature variations following the pressure stimulation (Fig. 3). These sex differences in vasomotor activity suggest a greater interaction between mechanisms related to sympathetic regulation of vasomotor responses and nociception, which is in line with previous studies showing sex differences in sympathetic nervous system regulation [16-18]. These sex differences have been attributed to sympathoadrenal processes regulating blood pressure and vascular resistance, although this relationship is not fully understood [41]. Interestingly, while greater temperature variations were seen in females compared to males in both sessions, females showed a higher number of upper limb regions affected by referred pain only at baseline (Table 2). Besides, it is necessary to take into account the aforementioned interpretation of the data in the context of the thermography margin of error, which leads to the conclusion that real changes only occurred in the female group. These differences might be related to females being more sensitive to mechanical pressure [42] and having a less efficient endogenous pain modulation than males [43]. Both factors can contribute to the larger referred pain areas found in females at baseline but not on Day 1, when the participants were assessed in a sensitized condition. In fact, original studies indicate that female facilitation of pain perception could be observed through signs of sympathetic activity such as pupil constriction [44]. Furthermore, it has been suggested that sex-differences in perceived muscle pain may be due to supraspinal rather than peripheral or spinal mechanisms [45]. This has further been supported in a study where the peripheral anesthetic block of nociceptive tissue afferents, after the injection of hypertonic saline, only prevented increases in skin blood flow in males [18]. Furthermore, the possibility of females showing higher temperature variations and more expanded pain, due to receiving a more intense exercise than males, can be ruled out, as no differences were found in perceived DOMS on Day 1 between females and males.

Taken together, these findings suggest that sympathetic vasomotor activity is likely related to the facilitated referred pain response found in females compared with males, and that sympathetic vasomotor activity may partly be accountable for the higher prevalence of clinical widespread pain in females [46]. Studies carried out in healthy individuals found either a decrease [15] or no changes [32] of temperature [47] when a chemical model of sensitization was used through infiltration of capsaicin [15] or glutamate [12,32]. In this study a decrease of temperature was found, although using a DOMS model instead of a chemical one, which the authors think clinical pain (prolonged movement-related pain) can be mimicked more successfully. In the case of studies regarding painful conditions, Skorupska et al. [34] found an increase in temperature following treatment of sore muscles in individuals with sciatica, but a decrease in temperature following treatment of individuals with sciatica and without sore muscles. Moreover, this study also showed a higher decrease of temperature in distal body regions, similarly to what the authors of the present study found, although the reason for this is unknown and should be explored in future studies.

The software used to analyze the infrared thermographic image did not allow for accurately superimposing the thermographic image with the area of referred pain. This has previously been considered important when assessing small regions with thermograms [48], as the results obtained tend to be more reliable. Additionally, pain drawings were done on a paper-body chart, but if an electronic body chart would have been used, it would have been possible to calculate the total area of self-perceived pain in a more detailed manner [49] than simply calculating the number of body regions affected by referred pain. Another limitation is that although the DOMS model can mimic clinical pain [50], the study was conducted in a small sample of healthy individuals, and therefore the results cannot be generalized or extrapolated to painful conditions. Moreover, the observational nature of this study and the absence of a control group and/or control side does not allow an evaluation of causality. Future studies should take into account these limitations and also include other outcome measures of sympathetic flow and vasomotor activity to better analyze potential associations with referred pain.

This is the first study to demonstrate temperature decreases in the area of experimental referred pain which correlate with the expansion of referred pain in a sensitized condition based on DOMS. Moreover, these findings seem to be more pronounced in females than males. Collectively, the study findings indicate that sympathetic vasomotor responses, as measured by infrared thermography, could be considered a valuable objective biomarker to assess pain conditions manifesting as expanded pain distribution.