Chocolate intake and muscle pain sensation: A randomized experimental study

Alexandra Hajati; Mario Brondani; Lina Angerstig; Victoria Klein; Linda Liljeblad; Essam Ahmed Al-Moraissi; Sofia Louca Jounger; Bruna Brondani; Nikolaos Christidis

doi:10.1371/journal.pone.0284769

. 2023 May 24;18(5):e0284769. doi: 10.1371/journal.pone.0284769

Chocolate intake and muscle pain sensation: A randomized experimental study

Alexandra Hajati ¹, Mario Brondani ², Lina Angerstig ¹, Victoria Klein ¹, Linda Liljeblad ¹, Essam Ahmed Al-Moraissi ³, Sofia Louca Jounger ¹, Bruna Brondani ², Nikolaos Christidis ^1,^*

Editor: Andrea Martinuzzi⁴

PMCID: PMC10208501 PMID: 37224109

Abstract

Background

Chocolate, as a cocoa-derived product rich in flavanols, has been used for medical and anti-inflammatory purposes. Therefore, the aim of this study was to investigate if the ingestion of different percentages of cocoa products affects the experimentally induced pain caused by intramuscular hypertonic saline injections in the masseter muscle of healthy men and women.

Methods

This experimental randomized, double-blind, and controlled study included 15 young, healthy, and pain-free men and 15 age-matched women and involved three visits with at least a 1-week washout. Pain was induced twice at each visit with intramuscular injections of 0.2 mL hypertonic saline (5%), before and after intake of one of the different chocolate types: white (30% cocoa content), milk (34% cocoa content), and dark (70% cocoa content). Pain duration, pain area, peak pain, and pressure pain threshold (PPT) were assessed every fifth minute after each injection, up until 30 min after the initial injection. Descriptive and inferential statistics were performed using IBM® SPSS (Version 27); significance level was set to p<0.05.

Results

This study showed that intake of chocolate, no matter the type, reduced the induced pain intensity significantly more than no intake of chocolate (p<0.05, Tukey test). There were no differences between the chocolate types. Further, men showed a significantly greater pain reduction than women after intake of white chocolate (p<0.05, Tukey test). No other differences between pain characteristics or sexes were revealed.

Conclusion

Intake of chocolate before a painful stimulus had a pain-reducing effect no matter the cocoa concentration. The results indicate that perhaps it is not the cocoa concentration (e.g., flavanols) alone that explains the positive effect on pain, but likely a combination of preference and taste-experience. Another possible explanation could be the composition of the chocolate, i.e. the concentration of the other ingredients such as sugar, soy, and vanilla.

ClinicalTrials.gov Identifier: NCT05378984.

Introduction

Pain is considered a global health issue since it not only influences quality of life for one-fifth of the world’s adult population, but also causes massive costs for patients, the health care system, and society [1, 2]. Luckily, most times pain will be transient, as it is associated with a lesion or disease that will heal. The word pain comes from a Greek word known as poine, which means penalty. In modern times, pain is described as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage” [3].

According to its temporal and aetiological categorization, pain can be classified as acute or chronic, and as nociceptive, neuropathic, nociplastic, idiopathic (e.g. pain of unknown origin), or mixed pain [4, 5]. Acute nociceptive pain is a sensation which arises when noxious peripheral stimuli activate the free nerve endings of nociceptive pathways. This stimulus does not need to induce tissue damage but has the intensity to reach the pain thresholds in order to elicit a pain sensation. Hence, acute pain is also a protective mechanism that helps prevent further injury by generating a reflex withdrawal [6, 7]. Chronic pain, on the other hand, is defined as pain that persists after healing and continues for at least 3 months [8].

To understand clinical pain, experimental pain models are utilized with healthy and homogenous participants, without confounding factors such as medications, age differences, illnesses, or external factors such as weather, ongoing wars. Furthermore, they can be performed in a standardized and controlled setting, with distinct start and end-points allowing quantitative assessments [9]. The use of hypertonic saline as the substance to chemically induce pain of muscular origin in the orofacial region in particular has been deemed valid [10–13]. Its acute nature and pronounced sensation of deep and diffuse pain, along with pain referral, is believed to mimic the sensation of patients with orofacial pain of muscular origin [11–17].

A common orofacial pain is associated with the temporomandibular disorder (TMD), described as all pain conditions affecting the masticatory muscles (and associated structures) and/or the temporomandibular joints [18]. Myalgia, i.e., pain in the jaw muscles, is the most common type of TMD, and leads to decreased mouth opening, pain when chewing or moving the jaw, soreness, and headaches [18]. Pain associated with TMD has a prevalence ranging from approximately 10 to 20% and is 1.5 to 2 times higher in women than men [13, 18–21]. The mechanisms behind why men are less predisposed to having TMD than women are still not clear [22].

It is suggested that TMD myalgia has a multifactorial aetiology due to complex interactions between biological, psychological, social, and environmental factors [23, 24]. When it comes to the biological factors, which are of interest here, some studies have suggested that micro-inflammation is involved in the development of TMD myalgia, where algogenic substances activate or sensitize nociceptive-free nerve endings, thereby eliciting a pain sensation [25–28]. One of these algogenic substances is the neurotransmitter serotonin, a target for many psychopharmaceutical drugs [29, 30]. Serotonin, which is found in blood platelets and in the central nervous system (CNS) [29], is released due to tissue damage or ischemia, as well as during inflammation; however, it has also been shown to regulate mood [31, 32]. Serotonin concentrations in patients with chronic myalgia have been shown to be significantly higher than those without [33].

Cocoa is believed to be linked to serotonin through tryptophan, a precursor of serotonin and an essential amino acid found in this widely consumed dietary product [34]. Cocoa has been used for medical and anti-inflammatory purposes throughout history, and it has been shown that cocoa-derived products rich in flavanols can reduce inflammation [35, 36].

It has also been found that a cocoa-enriched diet inhibits neurogenic inflammatory pain in rats, which implies the possible use of cocoa as an alternative therapy for pain control in humans [37]. There are indications that the type of chocolate (e.g., percentage of cocoa solids) plays an important role regarding its effect on sensory experiences [34]. However, the preference for a certain type of chocolate (e.g., sugar content, texture, and aroma) could also have an effect on pain based on a person’s psychological state or mood [34, 38–40] given that the composition of chocolate varies considerably [41].

Taken together, the aim of this study was to investigate if the ingestion of products with different percentages of cocoa affects the experimentally induced pain caused by intramuscular injections of hypertonic saline into the masseter muscle in healthy men and women. The hypothesis of the project was that the higher the cocoa content of the chocolate consumed, the less pain is experienced.

Materials and methods

The experiment took place between March 1 and December 20, 2020, at a research lab in the Department of Dental Medicine, Karolinska Institutet, Huddinge, Sweden. Ethics approval was obtained from the Swedish Ethical Review Authority (Dnr: 2019/05785), and the project followed the principles for medical research according to the declaration of Helsinki, as well as Good Clinical Practice (GCP) guidelines. Furthermore, the study was registered on ClinicalTrials.gov (Identifier: NCT05378984). The participants received both written and verbal information and gave their verbal and written consent.

Participants

The minimum sample size was estimated based on a normally distributed standard deviation of 30% [9, 29], a significance level (α) of 0.05 and a power (β) of 99%. In turn, 13 pairs of healthy participants were warranted. Considering the risk of dropouts, 15 men and 15 women were included and attended all three sessions without any dropouts, as shown in the CONSORT Flow Diagram (Fig 1). The power to detect a significant effect within the 15 pairs was also guaranteed at >0.999 (99%) using an effect (f) of 3, an error probability (α) of 0.05 and a correlation among the repeated 6 measures of 0.5.

Inclusion criteria for participating was men and women aged between 18–40 years in good general health. Exclusion criteria eliminated those with any pain-related diagnosis of TMD in the orofacial region, with headaches, systemic muscular or joint diseases (fibromyalgia or rheumatoid arthritis), whiplash-associated disorders, neurological disorders, psychiatric disorders, or allergies to any of the substances used. Recruitment of participants was done mainly through information to students at the Odontology Department at Karolinska Institutet in Huddinge, Sweden. Further information was given about the study, both in writing and verbally, to participants before the study began.

Study design

The study was designed as a randomized, controlled, and double-blind study. All participants completed questionnaires regarding their psychosocial status at the first visit prior to inclusion, and these included depression, somatization, anxiety, pain catastrophizing and stress (Axis II of the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD)) [18]. The following questionnaires were used to assess self-reported emotional functioning:

The Patient Health Questionnaire (PHQ-9): to assess self-reported depression symptoms and mental disorders including 9 items (each scored 0–3) based on the criteria for mental disorders in the DSM-IV diagnostic criteria for depressive disorders. The overall score is calculated into different levels of severity depending on the points: normal (0–4 points), mild (5–9 points), moderate (10–14 points), moderately severe (15–19 points), and severe (20–27 points) [42].
The Generalized Anxiety Disorder screener (GAD-7): to assess self-reported anxiety, consisting of 7 items (each graded 0–3) for assessing degree of anxiety related to pain. The total score according to severity is divided into: normal (0–4 points), mild (5–9 points), moderate (10–14 points), and severe (15–27 points) [43].
The Patient Health Questionnaire (PHQ-15): to assess self-reported somatization and nonspecific physical symptoms, consisting of 15 items (each graded 0–2) and divided from normal to severe nonspecific physical symptoms: normal (0–4 points), mild (5–9 points), moderate (10–14 points), and severe (15–30 points) [44].
The Pain Catastrophizing Scale (PCS-13): to assess self-reported feelings of rumination and magnification regarding pain, including 13 items (each scored 0–4) depending on the extent. The total score according to the risk of pain catastrophizing is divided into: normal (0–19 points), risk of pain catastrophizing (20–29 points), and high risk of pain catastrophizing (≥30 points) [45].
The Perceived Stress Scale (PSS-10): to assess the severity of stress including 10 items (each scored 0–4) depending on frequency of thoughts and feelings regarding stress during the last 30 days. The overall score according to the level of stress: normal level of stress (0–12 points), moderate level of stress (13–20 points), and severe level of stress (21–40 points) [46].

Prior to inclusion, all participants underwent examination of the orofacial region according to the DC/TMD [18] by a blinded examiner (AH–for all men; and VK–for all women).

1. Induction of experimental pain

During each experimental session (~ 1 hour), acute pain was induced by intramuscular injections (0.2 ml) of sterile hypertonic saline (58.5 mg/ml) into the most prominent point of the right side masseter muscle (assessed during contraction) by a non-blinded examiner (LA–for all men; and LL–for all women). Before injection, the skin was cleaned with a swab containing isopropyl alcohol (70%). To ensure intramuscular injection, a cannula of 19*0.4 mm was used and inserted perpendicular to the skin-surface covering the masseter muscle to a depth of 15 mm, as previously described by Christidis et al. [11]. Immediately after injection, assessments of pain characteristics and pressure pain thresholds (PPT) were performed. After 5 minutes pain had disappeared, but assessments continued for 30 minutes.

After the first 30 minutes of the experiment, including injection and assessments, one random piece of chocolate (3.6 g) was provided to the participant and 5 minutes later a second injection of hypertonic saline was given in the exact same manner as the first injection. All assessments were repeated in the same manner, as shown in Fig 2. Thus, each participant served as their own control (pre- or post-chocolate intake). This procedure has also been explained in detail in a study by Christidis et al. [11].

Fig 2 — At 0 minutes hypertonic saline is injected into the masseter muscle, every fifth minute PPT is assessed for 30 minutes, then this is repeated after intake of either white, milk, or dark chocolate, in healthy age-matched men and women (15 of each). DC/TMD = Diagnostic Criteria for Temporomandibular Disorders. VAS = Visual Analogue Scale (0–100). PPT = Pressure Pain Threshold.

2. Intake of chocolate

The chocolate given to the participants contained a different concentration of cocoa: 30% (white), 34% (milk), and 70% (dark), distributed in a randomized and double-blinded order. The participants were given the chocolate with their eyes closed. The non-blinded examiner (LA, LL) placed the chocolate in the participant’s mouth in each experiment, while the blinded examiner (AH, VK) did all the assessments. To ascertain that there was no possible carry-over effect, the different types of chocolate were given at three different visits to all participants with at least 1 week of washout between visits. The participants were asked to tell which of the three chocolates they preferred once they completed all three sessions.

To randomize the order in which the chocolates were given to each participant, an internet-based site was used (www.randomization.com; Seed 19525). The randomization was done in five blocks of six participants each, by a researcher who did not participate in the data collection.

The chocolates used in this study were packaged at Gem Chocolates®, Vancouver, Canada, using wafers from Belcolade manufacture in Belgium. They were all made uniform in shape and placed in homogeneous bags, making them blinded for participants and examiners. Each bag contained a single type of chocolate of a given concentration of cocoa. Each type of chocolate was made with a variation in the concentration of cocoa mass (cocoa nibs + cocoa butter), sugar, emulsifier, natural vanilla flavoring, and other products. Based on the US Food & Drug Administration (FDA), the chocolates were:

Dark chocolate: content of 70% cocoa (cocoa beans: 67.495%, cocoa butter: 3.2%, sugar: 28.3%, whole milk powder: 0.5%, sunflower lecithin: 0.4% and e476: 0.1% [emulsifiers], and natural vanilla extract: 0.005%),

Milk chocolate: content of 34% cocoa (cocoa beans: 9.8%, cocoa butter: 25.9%, sugar: 42.9%, whole milk powder: 21%, sunflower lecithin: 0.4% [emulsifier], and natural vanilla flavor: 0.04%),

White chocolate: content of 30% cocoa (cocoa butter: 30%, sugar: 43.16%, whole milk powder: 21.2%, dried skimmed milk: 5%, lecithin: 0.6% [emulsifier], natural vanilla flavor: 0.04%) [47].

The manufacturer also advises that their product may contain traces of soy. Of note, cocoa content comes from both the cocoa butter and the cocoa nibs that together make the cocoa mass (that is, the same 70% dark chocolate can have different percentages of cocoa nibs and cocoa butter and still be 70%).

3. Assessment of pain variables

The participants were requested to grade their perceived pain intensity continuously on a 0 to 100 mm Visual Analogue Scale (VAS), with 0 representing “no pain” and 100 representing “worst imaginable pain”. Pain intensity was marked as participants were prompted to do so every fifteenth second for 5 minutes (20 grades in total). At the end of the last grading, 5 minutes after injection, the participants were asked to mark the maximal subjective induced pain spread using a chart drawing with two lateral views of the head, one extraoral and one intraoral.

4. Assessment of pressure pain threshold (PPT)

PPT was assessed using an electronic pressure algometer (Somedic Sales AB, Hörby, Sweden) with a 1 cm² probe tip covered in 1 mm rubber on the skin surface of the masseter muscle. The pressure was applied at the most prominent point of the masseter muscle, coinciding with the site of the saline injection. Using a horizontal angle, the pressure was increased by the blinded examiner at a controlled rate of 30kPa/s. When pressure merged into pain, the subject was instructed to press a signal button. The PPTs were recorded one time every fifth minute after the injection for a total of 6 measurements within 30 minutes, i.e. 5, 10, 15, 20, 25, and 30 minutes after injection. The recording of PPT was only done once at each time point to reduce the risk of sensitization by repeated pressure stimuli. A standardized point was also chosen as the reference point on the left or right index fingertip on which the PPT was also assessed every fifth minute.

Data analyses

Statistical analyses were conducted on pain intensity, peak pain, pain duration, and pain area. Face charts marking the pain area were scanned using a Ricoh MP C6004ex printer with 300 dpi resolution, followed by using Adobe Photoshop CC2019 (Adobe Systems Incorporated USA) to mark the encirclements on the drawings and to express them in arbitrary units (au).

Statistical analyses

Statistical analysis was performed using SPSS 28 (SPSS Inc. Chicago, IL, USA). To test the normality of the data, the Shapiro-Wilk’s test was used. Mean and standard deviation (SD) were used for descriptive statistics when data was normally distributed, while median and interquartile range (IQR) were used for non-normally distributed data. Moreover, when data was normally distributed, parametric statistical methods were used, while non-parametric statistics were used for data that was not normally distributed.

The Kruskall-Wallis one-way analysis of variance (ANOVA) on ranks was used to test if there were any differences regarding background factors among the groups and different chocolate types.

The Shapiro-Wilk’s test indicated that data regarding pain intensity and PPT were normally distributed. Based on this, two-way repeated measures (RM) ANOVA was used to analyze differences over time, with chocolate type as the independent factor and time as the repeated factor. When the RM ANOVA indicated that there was a significant time difference, the Tukey test, for multiple comparison versus a control group (baseline), was used as a post-hoc test, as well as to test differences between chocolate types and interactions at different time points. Before statistical analysis of pain intensity and PPT, the values were normalized to baseline. Thus, the pre- and post-chocolate difference, i.e. the relative changes (%) were used in the statistical analyses.

Data regarding peak pain intensity, pain duration, and pain area from pain drawings were not normally distributed, not even after log transformation. Therefore, these data were analyzed using non-parametric methods. The Friedman RM ANOVA on ranks was used to test for differences between chocolate types. When a significant difference was indicated, the Dunn’s method for multiple comparisons versus a control group (baseline) was used as a post-hoc test. Since there were three different types of chocolate this was repeated three times, thus a Bonferroni correction was used resulting in a significance level of p<0.017 for this analysis. To test the effect of intake of low cocoa-content (30–34%) chocolate against high cocoa-content chocolate (70%) and to test for sex differences, the Mann-Whitney U-test was used.

The significance level was set to p<0.05 for all tests.

Results

Demographic data

Fifteen healthy and pain-free men with a mean (SD) age of 24.1 (3.4) years and 15 age-matched healthy and pain-free women with a mean (SD) age of 25.1 (2.6) years participated in this study.

The Kruskall-Wallis one-way ANOVA on ranks did not show any differences in psychosocial state between the different types of chocolate (Table 1). The scores for PHQ-9, PSS-10, PCS, GAD-7, and PHQ-15 were also similar.

Table 1. Baseline demographic status of 15 healthy, pain-free women and 15 healthy, pain-free age-matched men, i.e. before injection of any of the substances.

	Women (n = 15)	Men (n = 15)
Age
Mean (SD)	25.13 (2.588)	24.07 (3.515)
Min-max	21–31	19–34
Stress (PSS-10)
Median (IQR)	3 (4)	11 (11)
No stress (0–12 points)	n = 15	n = 10
Moderate degree of stress (13–20 points)	n = 0	n = 5
Severe degree of stress (21–40 pints)	n = 0	n = 0
Depression (PHQ-9)
Median (IQR)	5 (4)	3 (5)
Normal (0–4 points)	n = 7	n = 10
Mild (5–9 points)	n = 6	n = 5
Moderate (10–14 points)	n = 1	n = 0
Moderately severe (15–19 points)	n = 1	n = 0
Severe (20–24 points)	n = 0	n = 0
Pain catastrophizing (PCS)
Median (IQR)	5 (5)	2 (5)
None (0–19 pints)	n = 15	n = 15
Risk of clinical pain catastrophizing (20–29 points)	n = 0	n = 0
High risk of clinical pain catastrophizing (≥30 points)	n = 0	n = 0
Anxiety (GAD-7)
Median (IQR)	4 (5)	2 (4)
Normal (0–4 points)	n = 9	n = 13
Mild (5–9 points)	n = 3	n = 2
Moderate (10–14 points)	n = 1	n = 0
Severe (15–27 points)	n = 2	n = 0
Somatization (PHQ-15)
Median (IQR)	18 (6)	3 (3)
Normal (0–4 points)	n = 0	n = 11
Mild (5–9 points)	n = 0	n = 4
Moderate (10–14 points)	n = 1	n = 0
Severe (15–30 points)	n = 14	n = 0

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Data in the table are expressed as a median (interquartile range; IQR: 75th percentile minus 25th percentile). PSS-10 = Perceived Stress ScalePHQ-9 = Patient Health Questionnaire 9PCS = Pain Catastrophizing ScaleGAD-7 = Generalized Anxiety DisorderPHQ-15 = Patient Health Questionnaire 15

Milk chocolate was considered the favorite kind of chocolate for 27 out of 30 (14 men and 13 women) participants on a scale from 1 to 3, with a mean score of 2.73 points. White chocolate had a mean score of 2.40 points, while dark chocolate was the least favored, with a mean score of 1.87 points.

Pain intensity over time

White chocolate

The two-way RM ANOVA showed a significant time effect (F = 92.498; p<0.001), a significant difference with or without intake of white chocolate (F = 4.433; p = 0.044), and a significant interaction between time and intake of white chocolate (F = 1.782; p = 0.017). The post-hoc test showed that the experimentally induced pain intensity after intake of white chocolate was significantly lower than without intake 105–210 s after induction of pain (p<0.05, Tukey test), as shown in Fig 3.

Fig 3 — The mean (SEM) changes in experimentally induced pain intensity (VAS; visual analogue scale), by hypertonic saline injections, compared with baseline (BL) before and after intake of white, milk, and dark chocolate in 30 healthy, pain-free participants (A) and divided by sex into 15 women and 15 age-matched men (B-D). Assessments displayed were made every 15^th s beginning immediately after injection up to 300 s after injection. The changes in pain intensity are presented both for the entire group and for the sexes separately. *Significant difference compared to baseline after intake of white chocolate (Tukey test, p<0.05). #Significant difference compared to baseline after intake of milk chocolate (Tukey test, p<0.05). §Significant difference compared to baseline after intake of dark chocolate (Tukey test, p<0.05).

In men, the two-way RM ANOVA showed a significant time effect (F = 38.136; p<0.001), a significant difference with or without intake of white chocolate (F = 11.240; p = 0.005), and a significant interaction between time and intake of white chocolate (F = 2.576; p<0.001). The post-hoc test showed that the experimentally induced pain intensity after intake of white chocolate was significantly lower than without intake 60-240s after induction of pain (p<0.05, Tukey test), as shown in Fig 3.

In women, the two-way RM ANOVA showed a significant time effect (F = 56.698; p<0.001), but no difference with or without intake of white chocolate (F = 0.0483; p = 0.829), and no interaction between time and intake of white chocolate (F = 0.650; p = 0.880), as shown in Fig 3.

Milk chocolate

The two-way RM ANOVA showed a significant time effect (F = 103.081; p<0.001), a significant difference with or without intake of milk chocolate (F = 5.065; p = 0.032), but no significance on the interaction between time and intake of milk chocolate (F = 0.931; p = 0.550). Compared to white chocolate, the experimentally induced pain intensity after intake of milk chocolate was significantly lower than without intake (p = 0.032), as shown in Fig 3.

In men, the two-way RM ANOVA showed a significant time effect (F = 39.806; p<0.001), a significant difference with or without intake of milk chocolate (F = 4.927; p = 0.043), but no significance on the interaction between time and intake of milk chocolate (F = 1.059; p = 0.392). Compared to white chocolate, the experimentally induced pain intensity after intake of milk chocolate was significantly lower than without intake (p = 0.044), as shown in Fig 3.

In women, the two-way RM ANOVA showed a significant time effect (F = 66.499; p<0.001), but no significant difference with or without intake of milk chocolate (F = 0.863; p = 0.369); however, there was a significant interaction between time and intake of milk chocolate (F = 2.186; p = 0.002). The post-hoc test showed that the experimentally induced pain intensity after intake of milk chocolate was significantly lower than without intake at the 255 s time point after induction of pain (p<0.05, Tukey test), as shown in Fig 3.

Dark chocolate

The two-way RM ANOVA showed a significant time effect (F = 95.618; p<0.001), but no significant difference with or without intake of dark chocolate (F = 3.029; p = 0.092); however, there was a significant interaction between time and intake of dark chocolate (F = 2.209; p = 0.002). The post-hoc test showed that the experimentally induced pain intensity after intake of dark chocolate was significantly lower than without intake 105–165 s after induction of pain (p<0.05, Tukey test), as shown in Fig 3.

In men, the two-way RM ANOVA showed a significant time effect (F = 38.535; p<0.001), a significant difference with or without intake of dark chocolate (F = 6.058; p = 0.027), and a significant interaction between time and intake of dark chocolate (F = 2.556; p<0.001). The post-hoc test showed that the experimentally induced pain intensity after intake of dark chocolate was significantly lower than without intake 75–210 s after induction of pain (p<0.05, Tukey test), as shown in Fig 3.

In women, the two-way RM ANOVA showed a significant time effect (F = 62.151; p<0.001), but no significant difference with or without intake of dark chocolate (F = 0.0364; p = 0.852), and no significant interaction between time and intake of dark chocolate (F = 0.529; p = 0.957), as shown in Fig 3.

Sex differences

There were no sex differences at baseline in any of the assessments, i.e. before and after pre-treatment with any kind of chocolate. However, after intake of white chocolate the post-hoc test showed that men had a significantly greater reduction in pain intensity than women at 165–240 seconds after induction of pain (range of significance p = 0.002 to p = 0.049, Tukey test). There were no significant sex differences after intake of milk chocolate (p>0.348, Tukey test) or dark chocolate (p = 0.062, Tukey test).

Peak pain intensity

The peak pain intensity was not significantly affected by intake of any kind of chocolate (p>0.05). However, there was an overall reduction in intensity after intake of milk chocolate (6.5%) and white chocolate (3.2%), but not for dark chocolate (p = 0.204; Friedman RM ANOVA on ranks).

In men, intake of milk chocolate significantly reduced the peak pain intensity (p = 0.041; Friedman RM ANOVA on ranks); however, the reduction of peak pain intensity was not significant after white or dark chocolate intake. The intake of milk chocolate reduced the peak pain by 12.1%, while the reduction after intake of white chocolate and dark chocolate was 5.9% and 1.2%, respectively, although with no significant difference (p>0.05; Tukey test).

In women, intake of any of the types of chocolate did not affect the induced peak pain intensity (p>0.05). The intake of white chocolate did not affect the peak pain intensity whatsoever, while the intake of milk chocolate reduced the intensity by 1.1% and intake of dark chocolate increased peak pain intensity by 2.1% (p = 0.786; Friedman RM ANOVA on ranks).

When the sexes were compared, there was a significantly greater pain reducing effect after intake of milk chocolate for men than for women (p = 0.01), while there were no significant sex differences after intake of white or dark chocolate (p>0.05).

Pain duration

The duration of induced pain was not significantly affected by intake of any of the chocolate types (p>0.05), although it was reduced by 8.3% after intake of milk chocolate, by 14.4% after intake of white chocolate, and by 7.6% after intake of dark chocolate (p = 0.524; Friedman RM ANOVA on ranks).

In men, the duration of induced pain was not significantly affected by intake of any of the three chocolate types (p>0.05), although duration was reduced by 5.3% after intake of milk chocolate, by 21.1% after intake of white chocolate, and by 10.0% after intake of dark chocolate (p = 0.712; Friedman RM ANOVA on ranks).

In women, the duration of induced pain was also not significantly affected by intake of any of the chocolate types (p>0.05), although its duration was reduced by 25.0% after intake of milk chocolate and by 8.4% after intake of white chocolate, while it was unaffected after intake of dark chocolate (p = 0.127; Friedman RM ANOVA on ranks).

When the sexes were compared there was no significant difference in chocolate effect on pain duration after intake of any of the three chocolate types (p>0.05).

Induced pain spread

Intake of any of the chocolate types did not affect the induced pain spread statistically (p>0.05). In men, intake of any of the three chocolate types did not affect the induced pain spread (p>0.05). In women, intake of the chocolate types also did not affect the induced pain spread (p>0.05). There were no significant differences between sexes regarding chocolate effect on induced pain spread (p>0.05).

Pressure pain threshold (PPT)

White chocolate

The two-way RM ANOVA showed a significant time effect (F = 11.569; p<0.001), a difference with or without intake of white chocolate (F = 7.344; p = 0.011), and an interaction between time and intake of white chocolate (F = 5.677; p<0.001). The post-hoc test showed that the pressure pain threshold increased significantly after intake of white chocolate when compared to no intake 15 to 30 min after induction of pain (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed a significant time effect (F = 3.452; p = 0.006), no difference with or without intake of white chocolate (F = 0.00414; p = 0.949), and no interaction between time and intake of white chocolate (F = 0.201; p = 0.961). The post-hoc test showed a significant time effect at 30 min when compared to the baseline (p<0.05, Tukey test).

Fig 4 — The mean (SEM) percentage changes in pressure pain threshold (PPT; kPa), by hypertonic saline injections, compared with the baseline (BL) before and after intake of white, milk, and dark chocolate in 30 healthy, pain-free participants (A) and divided by sex into 15 women and 15 age-matched men (B-D). Assessments displayed were made every 15^th s beginning immediately after injection up to 300 s after injection. The changes in pain intensity are presented both for the entire group and for the sexes separately. *Significant difference compared to baseline after intake of white chocolate (Tukey test, p<0.05). #Significant difference compared to baseline after intake of milk chocolate (Tukey test, p<0.05). §Significant difference compared to baseline after intake of dark chocolate (Tukey test, p<0.05).

In men, the two-way RM ANOVA showed no time effect (F = 1.716; p = 0.142) and no difference with or without intake of white chocolate (F = 4.518; p = 0.052). However, there was an interaction between time and intake of white chocolate (F = 3.272; p = 0.010). The post-hoc test showed that the pressure pain threshold increased significantly after intake of white chocolate when compared to no intake 5 to 30 min after induction of pain (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed no time effect (F = 1.750; p = 0.135), no difference with or without intake of white chocolate (F = 1.451; p = 0.248), and no interaction between time and intake of white chocolate (F = 0.658; p = 0.657).

In women, the two-way RM ANOVA showed a significant time effect (F = 13.399; p<0.001) and no difference with or without intake of white chocolate (F = 2.5708; p = 0.122). However, there was an interaction between time and intake of white chocolate (F = 4.605; p = 0.001). The post-hoc test showed that the pressure pain threshold increased significantly after intake of white chocolate when compared to no intake 15 to 30 min after induction of pain (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed no time effect (F = 1.765; p = 0.131), no difference with or without intake of white chocolate (F = 1.209; p = 0.290), and no interaction between time and intake of white chocolate (F = 1.072; p = 0.383).

Milk chocolate

The two-way RM ANOVA showed a significant time effect (F = 10.165; p<0.001), no difference with or without intake of milk chocolate (F = 4.026; p = 0.054), and no interaction between time and intake of milk chocolate (F = 0.779; p<0.567). The post-hoc test showed a significant time effect at 10 to 30 min when compared to the baseline (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed no time effect (F = 2.301; p = 0.048), no difference with or without intake of milk chocolate (F = 0.656; p = 0.424), and no interaction between time and intake of milk chocolate (F = 0.933; p = 0.462).

In men, the two-way RM ANOVA showed no time effect (F = 0.753; p = 0.586). However, it showed a significant difference with or without intake of milk chocolate (F = 6.250; p = 0.025) and a significant interaction between time and intake of milk chocolate (F = 3.297; p = 0.010). The post-hoc test showed that the pressure pain threshold increased significantly after intake of milk chocolate when compared to no intake at the 15 and 30 min time points after induction of pain (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed no time effect (F = 0.806; p = 0.549), no difference with or without intake of milk chocolate (F = 1.282; p = 0.277), and no interaction between time and intake of milk chocolate (F = 1.023; p = 0.411).

In women, the two-way RM ANOVA showed a significant time effect (F = 19.355; p<0.001), no difference with or without intake of milk chocolate (F = 0.440; p = 0.518), and no interaction between time and intake of milk chocolate (F = 0.403; p = 0.845), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed a significant time effect (F = 3.764; p = 0.004), no difference with or without intake of milk chocolate (F = 0.379; p = 0.548), and no interaction between time and intake of milk chocolate (F = 0.466; p = 0.800).

Dark chocolate

The two-way RM ANOVA showed a significant time effect (F = 9.731; p<0.001), but no difference with or without intake of dark chocolate (F = 2.192; p = 0.150) and no interaction between time and intake of dark chocolate (F = 1.269; p = 0.280). The post-hoc test showed that the pressure pain threshold increased significantly after intake of dark chocolate when compared to no intake 15 to 30 min after induction of pain (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed a significant time effect (F = 3.254; p = 0.008), no difference with or without intake of dark chocolate (F = 0.102; p = 0.752), and no interaction between time and intake of dark chocolate (F = 0.513; p = 0.766). The post-hoc test showed a significant time effect at 15 to 20 min when compared to the baseline (p<0.05, Tukey test).

In men, the two-way RM ANOVA showed no time effect (F = 0.568; p = 0.725), no difference with or without intake of dark chocolate (F = 2.086; p = 0.171), and no interaction between time and intake of dark chocolate (F = 1.247; p = 0.297), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed no time effect (F = 2.351; p = 0.059), no difference with or without intake of dark chocolate (F = 1.090; p = 0.314), and no interaction between time and intake of dark chocolate (F = 1.025; p = 0.410).

In women, the two-way RM ANOVA showed a significant time effect (F = 15.040; p<0.001), no difference with or without intake of dark chocolate (F = 1.090; p = 0.314), and no interaction between time and intake of dark chocolate (F = 0.646; p = 0.665). The post-hoc test showed that the pressure pain threshold increased significantly after intake of dark chocolate when compared to no intake 10 to 30 min after induction of pain (p<0.05, Tukey test), as shown in Fig 4. Over the reference point, the two-way RM ANOVA showed no time effect (F = 2.481; p = 0.040), no difference with or without intake of dark chocolate (F = 0.196; p = 0.665), and no interaction between time and intake of dark chocolate (F = 0.192; p = 0.964).

Sex differences

There were no sex differences at baseline in any of the assessments, i.e. before and after pre-treatment with any type of chocolate. However, after intake of white chocolate the post-hoc test showed that men had a significantly greater increase in pressure pain threshold than women 5 min after intake, while women showed a significantly greater increase in pressure pain threshold than men 20 min after intake (p<0.05, Tukey test). When it comes to milk chocolate, women showed a significantly greater increase in pressure pain threshold than men 10 to 30 min after intake (p<0.05, Tukey test). Also, after intake of dark chocolate women showed a greater increase in pressure pain threshold than men 15 to 30 min after intake (p<0.05, Tukey test). Finally, there were no sex differences at any time point over the reference point regardless of chocolate type.

Discussion

The main finding was that all administrated types of chocolate, i.e., white (30% cocoa), milk (34% cocoa) and dark (70% cocoa) resulted in a reduction of the experimentally induced pain using the same experimental procedures as described in other studies [11, 48–50]. However, the cocoa content does not seem to be the main reason for reducing pain, disproving our hypothesis that the higher the cocoa content, the less pain experienced. Rather, the results indicate that it might be other ingredients in the chocolate such as sugar that explain these chocolate-induced reduction of pain variables.

In addition, PPT does not seem to be affected by intake of any type of chocolate, although this study could show some significant differences in PPT after intake of chocolate among either men or women. However, this is in line with previous studies which also have shown that PPT is not affected by various pain models and treatments [13, 51].

Function of cocoa

Previous studies have suggested chocolate as a complement to the traditional medical treatment of pain by inhibiting induced neurogenic inflammatory responses. However, these studies examined the effect of chocolate by using a neurogenic pain model on rats and not in intramuscularly induced pain in humans [36, 37]. In this present study, the pain reducing effect could not be fully attributed to the anti-inflammatory effects of cocoa, as found in a study showing that certain flavanols regulate the anti-inflammatory cytokine levels of IL-4 and TGF-β [36].

Cocoa-derived products that are rich in flavanols have been shown to reduce inflammation [36]. More specifically, flavonoids (type of antioxidant), which are found in certain fruits, vegetables, and in high concentrations in cocoa have been studied [34, 36, 52]. Also, as mentioned, intake of the amino acid tryptophan plays an important role in serotonin synthesis. However, daily intake of tryptophan needs to be at least six grams to have an increased effect on serotonin synthesis and subsequent mood effects [25], while this study used 3.6 g. Therefore, the amount of cocoa consumed during the experiments might be of importance to the outcome of the result. The small variations in pain experienced when comparing the different types of chocolate in this present study may have been due to the limited amount of chocolate that was consumed [36].

Differences between the chocolates and their sugar content

The sugar content in the different types of chocolate, mostly in the form of sucrose, has been shown to have an increased analgesic effect, and white chocolate tends to have a higher percentage of sugar than milk or dark chocolate. The increased effect might be due to the mechanism of release of opioids [53], with chocolate having a potentially similar mechanism of neurological addiction as other substances [54]. Given the potential effect of sugar content in chocolate on pain, both white and milk chocolate had a greater effect on reducing pain in the current study than dark chocolate which has a higher cocoa content. Milk chocolate also had a pain reducing effect regarding peak pain intensity and pain area.

This pain reducing effect is also in accordance with other studies [53, 55–57]. In these studies, the administration of sugar (e.g., sucrose, a disaccharide composed of glucose and fructose) was followed by a cold pressure test and the results showed an effect on pain tolerance and pain sensitivity [55]. A positive experience regarding the taste of different foods can stimulate mechanisms in the brain to release endorphins and neurotransmitters that result in increased pain tolerance [53]. The results of the chocolate ratings suggested the same, since white chocolate and milk chocolate had almost the same amount of “3” ratings, meaning they were preferred by the participants and both have higher amount of sugar than dark chocolate. And milk chocolate was the most pleasing according to the participants’ preferences, which might be one possible explanation as to why milk chocolate samples had a more significant effect on the pain intensity (but not duration) compared to white chocolate, even though these two consisted of a similar amount of cocoa.

But other studies found opposing results [36, 58]. However, these studies utilized a cocoa-enriched diet over a longer period of time, and one of them used a dose equivalent to a daily consumption of 33 g of cocoa powder for 14 days [58], which could also potentially affect the outcome compared to this study, which administered 3.6 g of chocolate per participant. The long-term ingestion of cocoa high in flavanols causes a change in platelet function similar to that of aspirin, becoming less potent [36, 59, 60], although this was not tested in the present study.

The fact that dark chocolate got less significant results may be because of the bitter taste, and less sugar content [53, 56]. Studies have reported significant differences in pain tolerance with sucrose water compared to cocoa infused solution, which showed no increase in pain tolerance [53]. This contrasts with the expectation that cocoa can be used for pain reducing effects [35, 36, 53].

Furthermore, in studies investigating the effect of chocolate on cold pain tolerance it was shown that preference, not cocoa concentration, was responsible for changes in tolerance [53]. Thus, based on the findings from the present study, and other studies [35, 36, 53], it would be intriguing to evaluate if a chocolate with a different balance of sweetness and cocoa concentration can affect pain tolerance more significantly than just sweet foods without any cocoa.

Study strengths and limitations

A significant strength of this study is that it was designed as a randomized double-blinded study, with both male and female participants within a small age-range. All the participants in the study fully completed the questionnaires and experiments, with no missing data.

Pain induction by hypertonic saline has been widely used in several studies due to its ability to mimic clinical acute muscle pain [11]. In this study, the induced saline injection caused a deep masseter muscle pain (63-70/100) of moderate intensity that spread to various other regions, such as the teeth; this is also shown by other studies using experimentally induced pain as a pain model [11, 51, 53]. Hence, another strength is that the induced pain in the present study had an intensity that can be considered as clinically relevant.

Despite its findings, this study is not without limitations. We did not control for any ingestion of sweet foods before the visit, which could have affected the results [53]. However, probable food intake by some participants before the visit should not have affected the general results, since participants act as their own control at each visit and the results were so similar between participants. Another limitation is the time after ingestion of chocolate. Perhaps the time after ingestion was not enough to achieve any flavanol modulation of neurogenic inflammation. We also did not control for the composition of the chocolate types, such as soy, or vanilla content, which indeed could have influenced the outcome, as results indicate that it might be dependent on the individual’s preference and taste-experience, or even other ingredients in the chocolate that could explain this chocolate-induced reduction of pain variables.

One important confounding factor is that once the participant has been given the chocolate and taste it, they likely know what type of chocolate they were eating. Their preference could interfere with results. On the other hand, in this study preference and taste-experience were included in the results, which might have helped to address the limitations mentioned above.

As mentioned before, another limitation was the amount of chocolate that was given to the participants (3.6 g), which is much lower than other studies exploring the effect of flavanol or tryptophan content [36, 40]. Lastly, the data presented here could have been analyzed using a full-scale crossover design, with a mixed-effect model with a period effect, and using other software like SAS (SAS Institute, Cary NC). Although we took advantage of a cross-over design by utilizing each subject as their own control and a smaller number of patients, the duration of the study was likely short compared to a full-scale design, and the washout period might not have been long enough to remove the effect from the different types of chocolate on one another. We did base our analytical choice on our previous studies using the same experimental methodology and also on several others using SPSS for the same study design [61]. Yet, we cannot claim that the chosen design and statistical analyses were infallible.

Future studies

Future studies using a cross-over design should investigate the significance of cocoa as a factor in pain experience, including the amount ingested and the duration of analgesic effects after ingestion. For example, chocolates similar in taste and sugar/sweetness content, but with different cocoa concentration (70% vs 30–34%), should be used to investigate the extent to which the cocoa itself influences the perceived pain. Lastly, studies should compare commercially available chocolate with the ones used in this study, as they differ in cocoa content, particularly the white chocolate and to some extent the milk chocolate.

Conclusion

This study showed that intake of any type of chocolate 5 minutes before a painful stimulus has a pain reducing effect no matter the cocoa concentration. The results indicate that perhaps it is not the cocoa concentration (e.g., flavanols) alone that explains the positive effect on pain, but likely a combination of preference and taste-experience. Another possible explanation could be the composition of the chocolate, i.e. the concentration of the other ingredients such as sugar, soy, and vanilla.

Supporting information

S1 Checklist

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S1 Data

(XLSX)

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S1 File

(DOCX)

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Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0284769.r001

Decision Letter 0

Andrea Martinuzzi

25 Nov 2022

PONE-D-22-13220Cocoa intake and muscle pain sensation: a randomized experimental studyPLOS ONE

Dear Dr. Christidis,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: *** General comments: ***

The design is a three-period three-treatment crossover design with the further complication of including a two-factor within-subject design with multiple evaluations over time in each period. This design requires special techniques to analyze correctly. See Senn (2002) for details about methodology. The current analysis is incorrect.

The manuscript lacks line numbers, therefore, it is difficult to provide specific suggestions. There are minor grammatic errors that might benefit from a third-party proof-reading.

In addition, it does not seem that the data are available for review.

*** Specific comments: ***

The authors presumably have already found that their design is not going to be handled correctly using SigmaPlot -- however, the repeated measures analysis that is proposed is incorrect. Also, it seems likely that the implied R-side correlation structure implemented by SigmaPlot is too simplistic. Further, the use of the Friedman test is even more limited in its ability to correctly analyze these data.

This crossover design features:

* 3 periods

* 3 treatments

* 6 sequences

* 30 subjects

The within period design features:

* 2 conditions (pre- and post-chocolate)

* Multiple time-points

* Multiple measures

While it is not clear that performing a correct analysis would lead to substantially different results, it is difficult to say ahead of actually performing it. A correct analysis will include treatment, sequence, subject, period, and carryover effects in the between-subject design, and pre/post and time in the within-subject design. It may be reasonable to omit the carryover, but this must be justified. Senn (2002) provides an excellent discussion of this issue that may be of help.

As mentioned in the limitations, there is most likely a period effect. Therefore, this must be accounted in the analysis at a minimum. The randomization restriction represented by the sequence is also important. A subject-level effect is required to produce the correct degrees of freedom for analysis (and potentially, depending on how the analysis is specified, a random effect of subject by period). Most likely, the authors would like to generalize their results past this specific set of subjects, so that subject should be considered as a random effect.

This design should probably be analyzed using either SAS or R. It is recommended to use SAS, unless the authors have access to expertise in using R to specify complex analyses. In SAS it would be feasible to implement the entire design as run using PROC MIXED. However, setting up the analysis does require a little experience, effort, and likely some trial and error and it would require both G-side and R-side specifications.

Having set this analysis up would provide several benefits. First, it allows all tests of interests to be performed as either omnibus tests or contrasts (whether corrected for multiple testing or not). Second, it allows covariates to be entered to evaluate their effects net of the treatment or vice versa. Third, it provides a template that correctly reflects the experiment design in all its complexity and can be used for all assessments (ideally).

Alternatively, while the fine-grained sampling over time is interesting, it seems to provide little benefit in the actual analysis. Therefore, a simpler method of analyzing the data would be as follows:

* Summarize the data across time using a statistic or statistics, e.g.:

- Area under the curve

- Peak value

- Other

* Determine the differential effect of chocolate using the summary statistic, e.g.:

- Post-chocolate minus pre-chocolate

- Ratio of post-chocolate to pre-chocolate

* Perform a standard 3-treatment 3-period crossover analysis

Some sample PROC MIXED code for this "simpler" analysis would be:

PROC MIXED;

CLASS SEQUENCE SUBJECT PERIOD TREATMENT;

MODEL DELTA = TREATMENT PERIOD;

RANDOM SUBJECT(SEQUENCE);

LSMEANS TREATMENT / PDIFF CL E;

RUN;

(Note that the code above could be extended to allow the pre/post x time design within subjects, and this would indeed be the starting point for that effort.)

A side benefit of this approach is that it allows an easier comparison of the various measures with each other via scatterplots or correlation analysis. It is also more likely to provide data that meet the assumptions of the linear model analysis.

The authors state that they evaluate normality of the data. This is a misnomer. One can easily see that in any case of interest with actual group differences that the data will not be normally distributed. What is assessed is the normality of the residuals from the linear model analysis. (Or, as in this case, suitably standardized or studentized residuals.)

Even if these residuals are not completely consistent with normality, it is usually preferable to use the same analysis structure throughout rather than a hodge-podge of statistical methods. Nonparametric analogues can be used as support for this. Recall that linear models still produce unbiased results even if error distributions are not normal. This pays dividends in interpretability and ease of representing the experimental design.

Note that the authors present significance tests based on ranks, but then quantitative results based on means. This is a common method to try to dodge the fact that rank-based methods provide no real quantitative statements but strikes one as disingenuous. Using a linear model approach is better for this purpose, even if the model fit is only fair.

*** Reference: ***

Senn, S. S. (2002). Cross-over trials in clinical research (Vol. 5). John Wiley & Sons.

Reviewer #2: This paper reports on a generally well-designed study producing novel data that intake of chocolate reduces acute pain induced by hypertonic saline injection into the masseter muscle of young healthy men and women. There are however several points that need to be satisfactorily addressed by the authors before it could be considered acceptable.

1. The first 4 paragraphs of the Introduction inexplicably focus on chronic pain, yet this study did not address chronic pain per se since it used an experimental model of acute pain which the Discussion states is used “to mimic clinical acute muscle pain”. The 1st part of the Introduction thus needs to be re-cast with a focus on acute pain and underlying mechanisms, including those expressed in the masticatory muscles.

2. The introduction’ s middle paragraph about cocoa inhibiting “trigeminal nerve cells……spinal cord” needs modification since trigeminal nerve cells predominantly project to the brainstem, not spinal cord.

3. There seems to be a disconnect in the description of the Study Design. Clearer wording is needed. Items in pain scores are said to have been graded on a 0-10 scale but this is followed by the statement that they were graded from 0 to 4.

4. Timelines are given in the Methods text and Flow Chart but It is not convincing that there was sufficient time for chocolate ingestion to have occurred to exert analgesic effects (eg, via modulation of neurogenic inflammation) especially during the initial time points where the pain reductions are extremely modest. Also the time designation on the Time axis in Figs 3 and 4 are tiny and very difficult for the reader with even 20/20 vision to discern.

5. The Study Design outlines measures to assess both pain intensity and pain disability, but the Results overlooks any presentation of the latter.

6. Strengths of the study included the use of different concentration of cocoa in the chocolate pieces ingested, and the finding that taste preference played a key role. The 1st couple of paragraphs of the Discussion discuss cocoa and its analgesic effects but it is unclear how a definitive conclusion could be reached from the present study that it is the cocoa in the ingested pieces of chocolate that was responsible for the modest analgesic effects. There were other ingredients in the chocolate (soy, sugar, vanilla) and there is no indication in the study design that these were controlled for, despite different cocoa concentration being used. Accordingly, the title of the paper should be modified to reflect “chocolate” not ‘cocoa’.

7. The !st sentence of the Discussion does not make sense; it seems to be missing words.

8. Likewise, the 1st sentence of the 9th paragraph about taste preference being “responsible for the response to cold pain tolerance” is unclear and needs to be re-formulated to make it clear how taste preference could affect a “response” to the “tolerance” of “cold pain”. The next 2 sentences also have a problem in the formatting (and show grammatical errors).

9. The Study Limitations will need to include those identified above (eg, Point 4).

Reviewer #3: The subject could be of great interest however, the design and the interpretation of the results are a limitation. The main concern is that the authors do not know or not show the composition of the chocolate. White chocolate has only cocoa fat with no flavanols at all (30% of cocoa fat is a lot really). They omit the sugar content in the different chocolates that should be around 50% for milk and white chocolate. Maybe these facts can better explain the preference and the un-expected results regarding the "higher flavanols the higher analgesic effect". I think that, unfortunately, it is not possible to get to any conclusion because there is no real control for the effect. It looks to me that it is the sugar content the one producing any effect and to probe it you would need a non -sugar content control

When authors say that is a controlled study: Which is the control?

The conclusion “The results indicate that it is not mainly the cocoa-concentration that has a positive effect on pain-characteristics, but the effect seems to be affected by the preference” is not based in the results. There is no significance or correlation to support it. Again I think the difference lays in the sugar content.

I don’t see the point of using an abbreviation for serotonin.

Did the authors check for a sequence effect in the assays (white, milk or dark) on the preference for one or other chocolate?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2023 May 24;18(5):e0284769. doi: 10.1371/journal.pone.0284769.r002

Author response to Decision Letter 0

13 Dec 2022

Response to reviewers:

PONE-D-22-13220

Cocoa intake and muscle pain sensation: a randomized experimental study

Nikolaos Christidis, 2022-12-04

Dear Academic Editor,

Thank you for considering this manuscript for publication. Since two of the reviewers approved the statistics and the third had some concerns, we have tried to address some parts of the statistics. The methods and set-up for this study have been used in several (hundreds) publications with hypertonic saline as experimental pain instigator, so any carry over effects have been taken into consideration in the study plan, thus not affecting the results. Furthermore, we have several publications regarding this model using the same statistics after consultation with a biomedical statistician, and this statistical method has also been approved in three doctoral theses from our group alone (Christidis Nikolaos 2010, Sofia Louca Jounger 2017, and Samaa Al Sayegh 2021). However, as it was suggested to use one statistical method for all analyses, we discussed using 2-way repeated measures for all analyses. Since data was not normally distributed we chose the more conservative non-parametric statistical Friedman repeated measures ANOVA method. In fact, we have already presented the pre-and post-chocolate effect difference for the analysis; we have also used the peak pain value for analysis, but since it was not obvious for the reviewer we have clarified that section. Finally, we do not have the statistical programs suggested by the reviewer, so we have to use SigmaPlot. Therefore, we hope that you still consider this manuscript based on the fact that the method used has been approved several times before.

We would like to thank all three reviewers for the comments and suggested changes in order to improve the quality of this manuscript. The changes are highlighted in yellow. We hope that this reply and changes in the manuscript are sufficient and will answer your queries.

Reviewer 1:

*** General comments: ***

The manuscript lacks line numbers, therefore, it is difficult to provide specific suggestions. There are minor grammatic errors that might benefit from a third-party proof-reading.

In addition, it does not seem that the data are available for review.

*** Specific comments: ***

This crossover design features:

* 3 periods

* 3 treatments

* 6 sequences

* 30 subjects

The within period design features:

* 2 conditions (pre- and post-chocolate)

* Multiple time-points

* Multiple measures

* Summarize the data across time using a statistic or statistics, e.g.:

- Area under the curve

- Peak value

- Other

* Determine the differential effect of chocolate using the summary statistic, e.g.:

- Post-chocolate minus pre-chocolate

- Ratio of post-chocolate to pre-chocolate

* Perform a standard 3-treatment 3-period crossover analysis

Some sample PROC MIXED code for this "simpler" analysis would be:

PROC MIXED;

CLASS SEQUENCE SUBJECT PERIOD TREATMENT;

MODEL DELTA = TREATMENT PERIOD;

RANDOM SUBJECT(SEQUENCE);

LSMEANS TREATMENT / PDIFF CL E;

RUN;

(Note that the code above could be extended to allow the pre/post x time design within subjects, and this would indeed be the starting point for that effort.)

*** Reference: ***

Senn, S. S. (2002). Cross-over trials in clinical research (Vol. 5). John Wiley & Sons.

Reply:

Thank you for reviewing this manuscript. We have added line-numbers in the version with the changes tracked.

When it comes to the concerns it seems that your major concern is the statistical method used. We have also been told by our biostatisticians to use non-parametric statistical methods for not normally distributed data as well as categorical and ranked data, even though they are more conservative. That is why we have used the Friedman repeated measures test for peak pain, pain duration and pain area.

Some of your concerns regard a carry-over effects. In this case each subject is its own control so we have used the pre- and post-chocolate difference as you suggested. However, since this was not obvious, we have clarified it in the intake of chocolate session as well as in the statistics section.

You also suggested that we should use simpler statistics such as the peak pain intensity difference, which already is presented in the results under its own section.

Further, there are thousands of publications on hypertonic saline as experimental setting and our group has used this method in several studies and in three theses that have been approved.

A final reason for not using more tests is that we do not wish to “significance-fish”.

Unfortunately, we do not have the statistical programs suggested, nor the knowledge to use the R system. Therefore, we use the analysis previously used and approved as suggested by our statisticians. We hope this response, where we consider your suggestion to use one method instead of mixing, where we have the pre- and post-chocolate difference, and the clarification in the text is acceptable for you.

Reviewer 2:

This paper reports on a generally well-designed study producing novel data that intake of chocolate reduces acute pain induced by hypertonic saline injection into the masseter muscle of young healthy men and women. There are however several points that need to be satisfactorily addressed by the authors before it could be considered acceptable.

Reply: Thank you for this comment. The first part of the introduction has changed focus to acute, experimental pain as suggested.

Reply: This has been corrected accordingly.

Reply: This part has been re-written to clarify any concerns.

4. Timelines are given in the Methods text and Flow Chart but It is not convincing that there was sufficient time for chocolate ingestion to have occurred to exert analgesic effects (eg, via modulation of neurogenic inflammation) especially during the initial time points where the pain reductions are extremely modest. Also, the time designation on the Time axis in Figs 3 and 4 are tiny and very difficult for the reader with even 20/20 vision to discern.

Reply: You are right; however, since this was the first study of its kind we did not know and chose to use the standard time-points for this experimental pain model. Perhaps time was not sufficient, but that requires a new study with different timelines. This is addressed in study limitations.

The figures might have been downsized in the system for the review. They have a high resolution of 900 dpi and can be zoomed in easily.

5. The Study Design outlines measures to assess both pain intensity and pain disability, but the Results overlooks any presentation of the latter.

Reply: This was misleading in the study design; no such assessment or analysis was performed since the study aimed at pain characteristics. This has been clarified.

Reply: The title has been changed accordingly

7. The 1st sentence of the Discussion does not make sense; it seems to be missing words.

Reply: This has been adjusted accordingly

Reply: This part has been adjusted as suggested.

9. The Study Limitations will need to include those identified above (eg, Point 4).

Reply: This has been added as you pointed out.

Reviewer 3:

The subject could be of great interest however, the design and the interpretation of the results are a limitation.

The main concern is that the authors do not know or not show the composition of the chocolate. White chocolate has only cocoa fat with no flavanols at all (30% of cocoa fat is a lot really). They omit the sugar content in the different chocolates that should be around 50% for milk and white chocolate. Maybe these facts can better explain the preference and the un-expected results regarding the "higher flavanols the higher analgesic effect". I think that, unfortunately, it is not possible to get to any conclusion because there is no real control for the effect. It looks to me that it is the sugar content the one producing any effect and to probe it you would need a non -sugar content control

Reply: We have added the composition of the chocolates and clarified in the first paragraph of the discussion that there are other, more probable factors that can explain the positive outcome, as suggested. This was also addressed in the study limitations and conclusion (as suggested further down).

When authors say that is a controlled study: Which is the control?

Reply: Each participant is its own control. First, they get an injection with hypertonic saline, then after half an hour chocolate, and 2 minutes later a new injection with hypertonic saline. Therefore, the control is pre-chocolate injection vs post-chocolate injection in each subject and the analysis is based on that. This has been clarified according to your question.

Reply: This has been adjusted as mentioned above.

I don’t see the point of using an abbreviation for serotonin.

Reply: This has been changed, as suggested.

Did the authors check for a sequence effect in the assays (white, milk or dark) on the preference for one or other chocolate?

Reply: Unfortunately, that data does not exist for one of the sexes, just which one they preferred. That is why we presented it in the “Demographic data”.

Attachment

Submitted filename: R1-Response to reviewers.docx

Click here for additional data file.^{(32.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0284769.r003

Decision Letter 1

Andrea Martinuzzi

3 Feb 2023

PONE-D-22-13220R1Chocolate intake and muscle pain sensation: a randomized experimental studyPLOS ONE

Dear Dr. Christidis,

The reviewers still highllight some major problems in the statistical analysis that critically undermine the validity of the work. Consider if you are able to address the issue otherwise consider withdrawal and possibly resumbission under different headline or journal.

Please submit your revised manuscript by MArch 30th. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Andrea Martinuzzi

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #2: The authors have satisfactorily addressed my remaining points from the previous review, except for point 7 since the first sentence still is unclear: how could it be that findings that “the pain reduction by the different types of chocolate presented in this study is line with previous studies “ (11, 48-50); none of these 4 cited studies investigated the effects of chocolate on pain. The sentence seems unnecessary and could be deleted unless the authors modify this sentence to clarify exactly what they mean by this unclear sentence.

Also note the revised wording on line 211 has introduced inappropriate tense changes in the same sentence; past tense should be used for both sets of verbs.

Reviewer #3: I think that the composition of the chocolates, now that it has been included, clearly shows that the effect, if any, is not due to the bioactives in cocoa, polyphenols and methylxanthines, but to maybe the cocoa fat and the sugar. In fact, the results show that in the case of men the sugar and fat content with no cocoa bioactives is more effective than the bioactive containing cocoa of dark chocolate.

I think that all this should be included in the abstract. It does not seem to be a question of preference but a question of sugar (and maybe fat contents)

This is in disagreement with most papers showing an effect of cocoa polyphenols and methylxanthines at different levels

See for instance:

Goya L, Kongor JE, de Pascual-Teresa S. From Cocoa to Chocolate: Effect of Processing on Flavanols and Methylxanthines and Their Mechanisms of Action. Int J Mol Sci. 2022;23(22):14365. Published 2022 Nov 18. doi:10.3390/ijms23221436

Gu Y, Lambert JD. Modulation of metabolic syndrome-related inflammation by cocoa. Mol Nutr Food Res. 2013;57(6):948-961. doi:10.1002/mnfr.201200837

etc

However, I think the paper should be published showing the results they got with really low amounts of cocoa solids (0% in white chocolate and only 9% in milk chocolate).

It is very important to show that this assay was done based on the bioactivity of cocoa (polyphenols and methylxanthines) however, the real content of this compounds, that was not determined, should be really low since cocoa fat has not beneficial effects that we knew until now.

Reviewer #4: The paper presents a randomized double-blinded controlled trial. First and foremost, the most important question if this is a powered trial to detect a significant effect. With 30 samples and without any power analysis this is not a powered trial. So any claim of significance based on p-value does not make sense.

Second, I reviewed the previous round of reviews and agree with the statistical reviewer that this should be analyzed using a crossover design. A mixed-effect model with a period effect should be used. The previous reviewer has given some excellent guidelines on how to analyze the data which I agree with. He/she also has SAS code, which was pretty tough. Unfortunately, all the suggestions are ignored for two reasoning. 1. They do not have access to SAS and 2. The authors have used similar simpler methods earlier which are accepted.

This is not a constructive way to address the reviewer’s suggestions. First, if SAS is not available, please use R, which is completely free to use. Second, just because previous papers are not reviewed by a statistician thoroughly it does not mean the methods used by them are all appropriate. It seems the investigative teams lack a statistician as otherwise converting the given SAS code to R and reanalyzing the data using a crossover design is not a big issue. The reviewer all mentions several other challenges that have to do with normality and model fitting which I cannot comment on given the suggestion is ignored.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2023 May 24;18(5):e0284769. doi: 10.1371/journal.pone.0284769.r004

Author response to Decision Letter 1

17 Feb 2023

Response to reviewers:

PONE-D-22-13220 R1

Cocoa intake and muscle pain sensation: a randomized experimental study

Thank you for further comments and suggestions to our manuscript.

Since some questions about the statistical analysis remain, we have tried to address them in the revised text.

Please note that by reviewing the entire manuscript, some of your suggested changes (with specific lines) might have shifted. We also took the opportunity to tie up the manuscript further.

Our point-by-point answer to your queries are presented in bold italics, ahead.

The changes are highlighted using the track changes function in Word. We hope that these changes answer too all your queries.

Review Comments to the Author

Reviewer #2:

The authors have satisfactorily addressed my remaining points from the previous review, except for point 7 since the first sentence still is unclear: how could it be that findings that “the pain reduction by the different types of chocolate presented in this study is line with previous studies “ (11, 48-50); none of these 4 cited studies investigated the effects of chocolate on pain. The sentence seems unnecessary and could be deleted unless the authors modify this sentence to clarify exactly what they mean by this unclear sentence.

R: Thank you for your comment, and sorry for the confusion. We were referring to the studies that used the same procedure (e.g., pain-induced in the masseter), not chocolate. We have edited that sentence accordingly.

Also note the revised wording on line 211 has introduced inappropriate tense changes in the same sentence; past tense should be used for both sets of verbs.

R: We are sorry for that oversight. We have corrected that sentence.

Reviewer #3:

I think that the composition of the chocolates, now that it has been included, clearly shows that the effect, if any, is not due to the bioactives in cocoa, polyphenols and methylxanthines, but to maybe the cocoa fat and the sugar. In fact, the results show that in the case of men the sugar and fat content with no cocoa bioactives is more effective than the bioactive containing cocoa of dark chocolate.

I think that all this should be included in the abstract. It does not seem to be a question of preference but a question of sugar (and maybe fat contents)

This is in disagreement with most papers showing an effect of cocoa polyphenols and methylxanthines at different levels

See for instance:

Gu Y, Lambert JD. Modulation of metabolic syndrome-related inflammation by cocoa. Mol Nutr Food Res. 2013;57(6):948-961. doi:10.1002/mnfr.201200837

etc

However, I think the paper should be published showing the results they got with really low amounts of cocoa solids (0% in white chocolate and only 9% in milk chocolate).

R: Thank you for your suggestion and support to our findings. We have now reworded the findings in both the abstract and the text to highlight that it was the sugar content (and low amounts of cocoa solids) rather than the cocoa content alone that influenced the pain sensation.

R: Thank you for your comment, appreciated. We performed power calculation with our statistician to confirm that we had a strong power (>0.999) to detected any difference, significantly.

R: We do welcome both reviewers’ comments and suggestions. We are sorry if we came across as dismissive of your concerns, that was not the intention. We were trying to explain that SPSS did the work that was aimed to be done in our study, and we (and others) have used before. We know it is not the only way to perform statistical analysis while recognising that SPSS (point-click based) has been used in trials of the same design by many other researchers – yet, it does not make it infallible compared to SAS (programme based). We consulted with out statistician to confirm our analysis and we have also added the discussion around limitation to address this issue. We also revised the entire manuscript to better adequate the language used to present and discuss the results in light of the potential limitations. Thank you very much for understanding.

Attachment

Submitted filename: R2-Response to reviewers.docx

Click here for additional data file.^{(25.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0284769.r005

Decision Letter 2

Andrea Martinuzzi

16 Mar 2023

PONE-D-22-13220R2Chocolate intake and muscle pain sensation: a randomized experimental studyPLOS ONE

Dear Dr. Christidis,

There are still major ponits to be addressed (see comments of reviewer 3).

Please submit your revised manuscript by April 20th. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Andrea Martinuzzi

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #5: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #3: No

Reviewer #5: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #5: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #5: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #5: No

**********

6. Review Comments to the Author

Reviewer #2: The authors have satisfactorily addressed the remaining points in my most recent review. However, I do not believe that the authors have satisfactorily addressed the point of Reviewer #3 (which was also related to a point that I raised in an earlier review) that sugar or other ingredients in the chocolate could have contributed to the reduction in pain and that this should be included in the Abstract. The revised Abstract now in its conclusion only refers to taste preference for sugar to explain the pain reduction, even though now the Discussion (lines 684-685) indicates that “other ingredients in the chocolate that could explain this chocolate-induced reduction of pain variables” and the Conclusion (lines 708-709) states that “Another possible explanation could be the composition of the chocolate, i.e. the concentration of the other ingredients such as sugar, soy, and vanilla. Comparable wording should be briefly reflected in the Abstract conclusion.

Reviewer #3: The abstract keeps being unclear and now I think that intentionally misleading. White chocolate might have 30% cocoa fat, but no flavanols at all (introduction and hypothesis is based on this). The results clearly show that if any the pleasure of fat plus sugar intake (that is what white chocolate and milk chocolate mainly provide) is the one producing the "analgesic" effect, very interestingly and in a statistically significant way in the case of white chocolate in men. I think that this needs to be said from the abstract.

Reviewer #5: The authors, in this round of revision, did their best to address the concerns raised. However, they relegated the suggestion of conducting a full-scale crossover design. It is not clear why they were reluctant to conduct it. I am OK to pass the paper, but more arguments are needed behind not using a cross-over design as a separate analysis.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #5: No

**********

PLoS One. 2023 May 24;18(5):e0284769. doi: 10.1371/journal.pone.0284769.r006

Author response to Decision Letter 2

20 Mar 2023

Response to reviewers:

PONE-D-22-13220 R2

Chocolate intake and muscle pain sensation: a randomized experimental study

Thank you for further comments and suggestions to improve our manuscript, in this case the abstract.

Our point-by-point answer to your queries are presented after the R:, ahead.

The changes are highlighted in yellow. We hope that this reply and changes in the manuscript are sufficient and will answer your queries.

Review Comments to the Author

Reviewer #2:

The authors have satisfactorily addressed the remaining points in my most recent review. However, I do not believe that the authors have satisfactorily addressed the point of Reviewer #3 (which was also related to a point that I raised in an earlier review) that sugar or other ingredients in the chocolate could have contributed to the reduction in pain and that this should be included in the Abstract. The revised Abstract now in its conclusion only refers to taste preference for sugar to explain the pain reduction, even though now the Discussion (lines 684-685) indicates that “other ingredients in the chocolate that could explain this chocolate-induced reduction of pain variables” and the Conclusion (lines 708-709) states that “Another possible explanation could be the composition of the chocolate, i.e. the concentration of the other ingredients such as sugar, soy, and vanilla. Comparable wording should be briefly reflected in the Abstract conclusion.

R: Thank you for your comment, and sorry for the confusion, that was not our intention. We have adjusted the conclusion making the wording comparable to the discussion and conclusion in the manuscript, as suggested.

Reviewer #3:

The abstract keeps being unclear and now I think that intentionally misleading. White chocolate might have 30% cocoa fat, but no flavanols at all (introduction and hypothesis is based on this). The results clearly show that if any the pleasure of fat plus sugar intake (that is what white chocolate and milk chocolate mainly provide) is the one producing the "analgesic" effect, very interestingly and in a statistically significant way in the case of white chocolate in men. I think that this needs to be said from the abstract.

R: Thank you for your observation. We now expanded a bit further on the reasoning of not utilizing a full-blown design. We did use some of its premises, including patients being their own control, and sing a smaller sample size. However, the duration of the study might have hindered a full-scale design. We have added this point into the limitations.

Attachment

Submitted filename: R3-Response to reviewers final.docx

Click here for additional data file.^{(24.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0284769.r007

Decision Letter 3

Andrea Martinuzzi

10 Apr 2023

Chocolate intake and muscle pain sensation: a randomized experimental study

PONE-D-22-13220R3

Dear Dr. Christidis,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Andrea Martinuzzi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

**********

6. Review Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: I thank you the authors for clarifying partially the real conclusion in this study. It is their paper and if they prefer to keep the reader in the confusion that flavanols might have something to do with any of this I won't ask for rejection. However, and this is my personal recommendation, I think that the authors should know and let the readers know that there should be 0 flavanols in white chocolate, the cocoa % correspond to cocoa fat and there are no flavanols in cocoa fat. Additionally in the methodology they could give an idea of the flavanol content expected in these kind of product, that should be 0 for white chocolate and really low for the milk chocolate. I think that talking about cocoa content when the content is of cocoa fat is not rigorous.

In my opinion, in fact, the design of the study (the product chosen for the study) is not correct for the hypothesis. Maybe the authors should say so as a limitation of their study.

Reviewer #5: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #5: No

**********

PLoS One. doi: 10.1371/journal.pone.0284769.r008

Acceptance letter

Andrea Martinuzzi

27 Apr 2023

PONE-D-22-13220R3

Chocolate intake and muscle pain sensation: a randomized experimental study

Dear Dr. Christidis:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Andrea Martinuzzi

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Checklist

(DOC)

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S1 Data

(XLSX)

Click here for additional data file.^{(58KB, xlsx)}

S1 File

(DOCX)

Click here for additional data file.^{(22.8KB, docx)}

Attachment

Submitted filename: R1-Response to reviewers.docx

Click here for additional data file.^{(32.6KB, docx)}

Attachment

Submitted filename: R2-Response to reviewers.docx

Click here for additional data file.^{(25.9KB, docx)}

Attachment

Submitted filename: R3-Response to reviewers final.docx

Click here for additional data file.^{(24.9KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Chocolate intake and muscle pain sensation: A randomized experimental study

Alexandra Hajati

Mario Brondani

Lina Angerstig

Victoria Klein

Linda Liljeblad

Essam Ahmed Al-Moraissi

Sofia Louca Jounger

Bruna Brondani

Nikolaos Christidis

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Participants

Fig 1. The CONSORT flow diagram.

Study design

1. Induction of experimental pain

Fig 2. Study design flowchart.

2. Intake of chocolate

3. Assessment of pain variables

4. Assessment of pressure pain threshold (PPT)

Data analyses

Statistical analyses

Results

Demographic data

Table 1. Baseline demographic status of 15 healthy, pain-free women and 15 healthy, pain-free age-matched men, i.e. before injection of any of the substances.

Pain intensity over time

White chocolate

Fig 3. Changes in pain intensity.

Milk chocolate

Dark chocolate

Sex differences

Peak pain intensity

Pain duration

Induced pain spread

Pressure pain threshold (PPT)

White chocolate

Fig 4. Changes in pressure pain threshold.

Milk chocolate

Dark chocolate

Sex differences

Discussion

Function of cocoa

Differences between the chocolates and their sugar content

Study strengths and limitations

Future studies

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Andrea Martinuzzi

Roles

Author response to Decision Letter 0

Decision Letter 1

Andrea Martinuzzi

Roles

Author response to Decision Letter 1

Decision Letter 2

Andrea Martinuzzi

Roles

Author response to Decision Letter 2

Decision Letter 3

Andrea Martinuzzi

Roles

Acceptance letter

Andrea Martinuzzi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK