Methyl salicylate, the main organic compound of natural wintergreen oil, is extracted from the leaves of the wintergreen plant (lat. Gaultheriae Aetheroleum) (Ojha et al., 2022). It is commonly used in topical applications such as gels, creams or patches (Yeoh and Goh, 2022). Topical formulations containing methyl salicylate have been shown to be effective in the treatment of muscle strain (Higashi et al., 2010), temporomandibular joint and masseter muscle pain (Lobo et al., 2004). Based on the principle of counterirritation, methyl salicylate has been suggested to have therapeutical effects by triggering irritation or cutaneous pain to mitigate pain of subdermal origin. However, the precise underlying physiological mechanisms are not fully understood (Gammon and Starr, 1941; Green, 1991; Barkin, 2013).

Following application, methyl salicylate rapidly penetrates the skin. In the epidermis and dermis, methyl salicylate undergoes hydrolysis, leading to the formation of active salicylic acid (Martin et al., 2004). Topical salicylates are considered to act primarily as rubefacients (Derry et al., 2014). Accordingly, several studies have recognized methyl salicylate as a vasoactive compound that increases local blood flow and raises skin or tissue temperature (Higashi et al., 2010; Dölen et al., 2015; Petrofsky et al., 2016; Anderson et al., 2017; Wang et al., 2022). Commercially available methyl salicylate products indicated for anti-inflammatory or analgesic purposes mostly contain other active compounds, such as menthol or camphor (Yeoh and Goh, 2022). Given the frequent co-formulation of methyl salicylate with other ingredients, it remains unclear whether the purported vasodilatory effects attributed to methyl salicylate (Higashi et al., 2010; Dölen et al., 2015; Petrofsky et al., 2016; Wang et al., 2022) are due to its individual effects or whether they are primarily mediated by other active compounds. Investigating the vascular effects of methyl salicylate alone may provide insight into its potential mechanisms of action. Moreover, direct penetration of salicylic acid has been shown to be dominant only to a depth of 3–4 mm (Singh and Roberts, 1993) and only small amounts are absorbed systemically (Martin et al., 2004). This suggests that topical methyl salicylate may act as a vasodilator in the skin, but not in the skeletal muscle tissue.

Topical methyl salicylate induces a characteristic thermosensory profile. While methyl salicylate products such as Perskindol^® Dolo Gel promise simply a comfortable warming sensation (Verfora, 2022), methyl salicylate induces also sensations of burning, stinging/pricking, and even cold, as opposed to just warmth (Green and Flammer, 1989; Green, 1991). In addition, methyl salicylate enhances the perception of warming and produces hyperalgesia to heating (Green and Flammer, 1989). The distinct thermosensory responses to topical methyl salicylate may be due to pharmacological manipulation of specific thermosensitive nonselective cation transient receptor potential (TRP) channels (Caterina et al., 1997; McKemy et al., 2002; Calixto et al., 2005; Bandell et al., 2007; Levine and Alessandri-Haber, 2007). However, it remains unclear, how the vascular and thermosensory responses to topical methyl salicylate relate over time.

Considering that (1) skin permeation of chemical agents is influenced by age, skin type and sex hormones due to small differences in the chemical composition of the stratum corneum (Souto et al., 2022), and (2) since this is the first study to investigate the vascular effects of methyl salicylate alone, the aim was to examine the effects in a relatively homogenous population. Therefore, the primary aim was to investigate the effects of the topical methyl salicylate on skin temperature (T_skin), skin microcirculation (MC_skin) and muscle oxygen saturation (SmO₂) compared to a placebo gel in healthy women. The secondary aim was to assess thermosensory responses (thermal sensation, thermal comfort) and to explore to which extent these sensations correspond to the physiological responses over time. It was hypothesized that (1) topical methyl salicylate would act as a cutaneous vasoactive compound, resulting in increased MC_skin but not SmO₂; (2) thermal sensations would correspond to changes in T_skin and follow a similar time course to the changes observed in MC_skin and (3) thermal comfort would be predominantly rated as comfortable.

T_skin, MC_skin and SmO₂ results are shown in Figure 1. Complete repeated measures ANOVA results and post hoc pairwise comparisons are presented in Supplementary Tables S1-S3.

This study aimed to investigate the effects of topical methyl salicylate on T_skin, MC_skin, and SmO₂ whilst simultaneously exploring its effects on thermosensation. The findings demonstrate an immediate, non-persisting increase in MC_skin, but not in SmO₂. Thermal sensation responses to topical methyl salicylate ranged from “cool” to “hot”, with more intense sensations reported at 5min post-application.

Following application, both gels caused a significant reduction in T_skin with minimal temperatures after 5 min (methyl salicylate: Δ-3.36°C, placebo: Δ-3.90°C). These results are consistent with previous studies demonstrating an immediate skin-cooling effect of topical gels in general, with mean/median T_skin reductions ranging from −2.1°C to −6.2°C (Hug et al., 2012; Lasanen et al., 2016; Petrofsky et al., 2016; Hunter et al., 2018). For topical menthol applications, it has been found that the cooling effect was not dependent on its concentration (Lasanen et al., 2016) and was found to be comparable to that of a placebo gel (Hunter et al., 2018). Therefore, the immediate cooling effect on tissue/organ temperature was not primarily attributed to the action of the active compound itself, but rather to the evaporation of water and/or alcohol from the skin surface, similar to the mechanism of perspiration (Lasanen et al., 2016; Hunter et al., 2018). The extent of the cooling effect may vary depending on the evaporative properties of the carrier substance, which differ between studies (8.0% (Lasanen et al., 2016) and 35–40% ethanol (Hunter et al., 2018), or isopropyl/propylene glycol (Hug et al., 2012)). In the present study, the carrier substance that included propylene glycol probably resulted in a less pronounced reduction in T_skin.

Methyl salicylate induced an immediate increase in MC_skin, peaking at 5 min, and remaining elevated at 10 min post-application, followed by a gradual decline thereafter. In contrast, the placebo gel resulted in consistently reduced MC_skin levels throughout the measurement period, with significantly lower levels compared to methyl salicylate. The significant changes observed in MC_skin suggest a short-term cutaneous vasodilatory effect of methyl salicylate and provide evidence for the claimed effects of a rubefacient. Many studies have recognized methyl salicylate as a vasoactive ingredient in topical formulations (Higashi et al., 2010; Dölen et al., 2015; Petrofsky et al., 2016; Anderson et al., 2017; Wang et al., 2022). However, given the common co-formulation of methyl salicylate with other ingredients, to date, no evidence was available to confirm the vasodilatory effect of topical formulations containing only this active ingredient.

After the initial drop in T_skin, both gels resulted in a gradual recovery in T_skin over time, with methyl salicylate showing significantly higher temperatures than the placebo gel between 5–40 min post-application. Regulation of local skin blood flow is an essential thermoregulatory mechanism: An increase in MC_skin through cutaneous vasodilation generally results in heat dissipation (Tan and Knight, 2018). Therefore, the immediate vasodilatory effect of methyl salicylate may have prevented a further decrease in T_skin, and subsequently accelerated the increase in T_skin. Nonetheless, given that after 45 min post-application, T_skin was still lower than the BL levels for both gels, indicates a sustained skin-cooling effect of gel evaporation.

While methyl salicylate demonstrated a transient increase in MC_skin, reflecting altered skin blood flow at a maximum depth of 1 mm (Limited, 2012), it did not induce a concomitant or delayed increase in blood flow within the superficial skeletal muscle (at approximately 15 mm depth (Ferrari et al., 2004)). Changes in SmO₂ levels over time were minimal for both gels, and no significant difference was found between the gels. This finding suggests that topical methyl salicylate does not enhance skeletal muscle blood flow. Topical products are widely used for the treatment of acute injuries and chronic musculoskeletal conditions (Derry et al., 2014; Derry et al., 2017). Many over-the-counter products are based on natural ingredients including wintergreen oil, menthol, rosemary, capsaicin, or camphor (Morton and Hall, 2002; Barkin, 2013; Kopustinskiene et al., 2022). To date, despite the widespread use of natural compounds and the interest in their therapeutic effects for musculoskeletal conditions, there is limited knowledge of their effects on skeletal muscle blood flow or SmO₂. One study reported that topical menthol induced blood flow in the quadriceps muscle (Petrofsky et al., 2016). However, the total measurement period (120 min) and the selected interval durations (15 min) may have been too long, considering that menthol is already cleared after 60 min post-application (Craighead et al., 2017). When measured over a measurement period of 50 min, menthol and camphor separately increased SmO₂ in the forearm, indicating enhanced local perfusion in skin and superficial muscle (Kotaka et al., 2014). Methyl salicylate, menthol and camphor undergo passive diffusion through the epidermis, reach the dermal microvascular networks and are absorbed systemically in detectable but low doses (Martin et al., 2004). Given such low doses, it remains questionable, as to whether, or to what extent, these topical chemicals have vasodilatory effects in the deeper tissues in general (Silva, 2020).

Subjective thermal sensation responses did not entirely align with the objectively measured T_skin. Following application, the initial decrease in T_skin was not clearly perceived with either gel. For methyl salicylate, at T_skin(min) measured after 5 min post-application, participants tended to experience either cold or warm sensations, with a lower percentage reporting a neutral sensation (4.8% vs. 57.1%). While thermal comfort was predominantly reported as comfortable for both gels, a notable proportion experienced slight thermal discomfort (42.9%) or discomfort (4.8%) after 5 min post-application with methyl salicylate. Over the subsequent measurement period, reaching T_skin(max) at 45 min, the intensity of these more extreme sensations gradually decreased, with an increasing percentage of participants reporting a neutral sensation similar to placebo. These findings indicate that topical methyl salicylate (12.9%), induced a generally more pronounced and varied thermosensory response within the initial 5 min, which aligns well with the peak in MC_skin observed at the same time point. Consistent with these observations, topical methyl salicylate was previously demonstrated to be reliably detectable at concentrations between 3-12%, with a perceived sensation intensity peak between 5–9 min post-application (Green and Flammer, 1989).

Methyl salicylate products such as Perskindol Dolo^® are commonly referred to as “hot gels” and are marketed with the implicit or explicit promise of inducing a comfortable and warming sensation (Verfora, 2022). However, the actual thermal sensation responses among participants were more varied and did not clearly evoke a clear cold or warm sensation. This discrepancy supports previous findings that methyl salicylate does not primarily induce a warming sensation (Green and Flammer, 1989) and highlights the complexity of individual sensory responses to topical methyl salicylate. Interestingly, methyl salicylate has been demonstrated to elicit sensation qualities that are independent of temperature, such as burning and stinging/pricking (Green and Flammer, 1989; Green, 1991). Participants were specifically asked to rate thermal sensation using a forced-choice scale, which required them to provide a response even if the sensation experienced was not primarily thermal in nature. This may have introduced a response bias, as the burning sensation evoked by methyl salicylate may have been understood as synonymous with a warm or hot sensation. Therefore, it is important to consider the possibility that participants’ interpretation of the sensations experienced may have been influenced by the presence of burning sensations associated with gel application.

The short-term cutaneous vasodilatory and distinct thermosensory effects of methyl salicylate observed in this study may be based on the short-term activation or inhibition of specific thermosensitive TRP receptors, such as it is suggested for menthol (Macpherson et al., 2006; Craighead et al., 2017; Hunter et al., 2018; Silva, 2020) or camphor (Moqrich et al., 2005; Xu et al., 2005; Selescu et al., 2013; Kotaka et al., 2014). Specifically, methyl salicylate has been shown to have both stimulatory and inhibitory effects on TRPV1 (vanilloid) (Calixto et al., 2005; Ohta et al., 2009), which responds to painful stimuli and heat (>42°C) (Caterina et al., 1997; Bandell et al., 2007), and to activate TRPM8 (melastatin) (Calixto et al., 2005) and TRPA1 (ankyrin) channels (Calixto et al., 2005; Levine and Alessandri-Haber, 2007), which are sensitive to cold temperatures (McKemy et al., 2002) (<25°C and <17°C respectively) (Bandell et al., 2007). In this study, both gels were stored at room temperature (23.3 ± 0.9 °C), which implies that the application of the gels was likely to initially activate TRPM8. Additionally for the methyl salicylate gel, distinct modulation of TRPV1, TRPM8 and TRPA1 (Calixto et al., 2005; Levine and Alessandri-Haber, 2007; Ohta et al., 2009), may have contributed to the varied thermal sensation response. To note, methyl salicylate induced a transient MC_skin peak and changes in thermosensation occurred mainly after 5 min post-application. In contrast, the vascular and thermosensory effects of menthol and camphor seem to last remarkably longer (menthol: elevated MC_skin between 15–45 min, cooling sensations 5–60 min post-application (Craighead et al., 2017); camphor: elevated MC_skin for 50 min and thermal sensations for 30 min respectively (Kotaka et al., 2014)). This suggests that the presumed action of methyl salicylate, menthol and camphor on thermosensitive TRP channels may differ in timing and intensity.

This study aimed to create a realistic therapeutic setting while maintaining a standardized placebo-controlled experiment. As such, the findings are provided considering several limitations and must be interpreted with caution. The protocol ensured consistent use of the same amount of gel, employing a uniform application technique and subjective verification of application pressure and motion. However, the conditions were not completely identical. The (1) variability in the skin penetration endpoint (T0) as well as (2) potential variations in skin humidity and chemical absorption may have introduced slight variability among participants. (3) Moreover, potential interaction effects between thermosensory perception induced by each gel on the left and right side of the spine cannot be excluded. Nevertheless, the within-subject design is likely to outweigh these variations. (4) Thermosensation was determined using slightly adapted standardized subjective ratings for thermal sensation and thermal comfort (Fabbri, 2015). (5) NIRS technology required coverage of the skin areas at each measurement time point, which might have had an occlusive effect of a few seconds on the skin. In light with these methodological limitations, similar studies may benefit from to further standardizing and refining the specific time points related to gel application (e.g., gel exit from the tube, initiation of gel application, completion of gel distribution, and subjective determination of skin penetration endpoint). Furthermore, future studies could consider measuring the temperature of each gel and the fingertips of the assessors applying the gel prior to application. This additional information could enhance the interpretation of T_skin results.

Further research is needed to improve our understanding of the effects of natural topical products on deeper laying tissues. Furthermore, it would be valuable to compare the effects of natural wintergreen oil and synthetic methyl salicylate (Ojha et al., 2022). Additionally, alternative delivery forms of methyl salicylate (e.g., patches, sprays) (Yeoh and Goh, 2022), through application during exercise and/or heat exposure (Danon et al., 1986) and/or higher methyl salicylate concentrations could be investigated to optimize skin permeation and improve its efficacy. Specifically for topical methyl salicylate, this study advises the assessment of sensation qualities that are independent of temperature (e.g., burning, stinging, and pricking). Methodologically strong studies are encouraged to thoroughly investigate individual and combined effects of natural compounds, such as menthol and camphor that act on thermosensitive TRP channels. This would contribute to a better understanding of their underlying mechanisms and potential synergistic or additive effects. Given the affordability of topical methyl salicylate, it would be worthwhile to explore its physiological, subjective, and presumed analgesic effects in musculoskeletal conditions. This research could provide valuable insights into its therapeutic potential.

To conclude, this study used advanced non-invasive techniques to investigate the vascular effects of topical methyl salicylate. By including thermosensation assessments, it could be explored how vascular and thermosensory responses relate over time. The findings indicate that topical methyl salicylate induces short-term cutaneous vasodilation, but it may not enhance skeletal muscle blood flow. In addition, this study highlights the complex sensory responses to its application, which may be based on the short-term modulation of thermosensitive TRP channels. This study provides insights for formulators seeking to develop effective topical applications with specific vascular and thermosensory responses, as well as for healthcare professionals advising on their use.