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\begin{document}

\title{Orthostatic stress testing in myalgic encephalomyelitis/chronic fatigue
syndrome (ME/CFS) patients with or without concomitant fibromyalgia:
effects on pressure pain thresholds and temporal summation}



\author[1]{C (Linda) van Campen}%
\author[2]{Peter Rowe}%
\author[3]{Freek Verheugt}%
\author[1]{Frans Visser}%
\affil[1]{Stichting Cardiozorg}%
\affil[2]{Johns Hopkins University}%
\affil[3]{OLVG}%


\vspace{-1em}



  
  \date{May 10, 2024}


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\selectlanguage{english}
\begin{abstract}
Introduction: muscle pain/fibromyalgia (FM) is common among individuals
with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We
recently demonstrated that during orthostatic stress testing, adults
with ME/CFS reported increased pain. In the current study, we
hypothesized that pain pressure thresholds (PPT) would decrease and
temporal summation (windup) would increase after head-up tilt testing
(HUT), and that the presence of co-morbid FM would be associated with
greater change in both measures. Methods and Results: 248 ME/CFS
patients (164 with FM and84 without FM), and 22 healthy controls (HC)
were analyzed. In HC there were no significant differences in PPT
between pre- and post-HUT (finger: from 4.7(1.6) to 4.4(1.5); shoulder:
from 2.8(1.0) to 2.9(1.0)). In ME/CFS patients with and without FM, a
significant decrease in PPT post-HUT was found compared to HC (both
p\textless{}0.0001). Patients with FM had a lower PPT pre- and post-HUT
(finger: from 2.0(0.9) to 1.5(0.8); shoulder: from 1.2(0.5) to 1.0(0.5)
compared to patients without FM (finger: from 5.0(1.6) to 3.3(1.5);
shoulder: from 2.2(0.9) to1.9(1.0) (p ranging from 0.001 to
\textless{}0.0001). In contrast to HC in ME/CFS patients windup was
increased compared to HC pre-HUT (both p\textless{}0.0001), but did not
significantly change post-HUT. Conclusions: Pressure pain threshold
decreased in ME/CFS patients with or without fibromyalgia after head-up
tilt test (HUT), compared to healthy controls. Windup pre-and post-HUT
was significantly higher compared to healthy controls, but did not
change from pre- to post-HUT. These results demonstrate that, like
exercise, orthostatic stress can negatively influence the physiology of
pain perception in ME/CFS.%
\end{abstract}%



\sloppy


\textbf{Orthostatic stress testing in myalgic encephalomyelitis/chronic
fatigue syndrome (ME/CFS) patients with or without concomitant
fibromyalgia: effects on pressure pain thresholds and temporal
summation}

C. (Linda) M.C. van Campen, MD\textsuperscript{1}

Peter C. Rowe, MD\textsuperscript{2}

Freek W.A. Verheugt, MD, PhD\textsuperscript{3}

Frans C. Visser, MD, PhD\textsuperscript{1}

1 Stichting CardioZorg, Planetenweg 5, 2132 HN Hoofddorp, Netherlands

2 Department of Paediatrics, Johns Hopkins University School of
Medicine, Baltimore, MD, USA

3 Onze Lieve Vrouwe Gasthuis (OLVG), Oosterpark 9, 1091 AC, Amsterdam,
Netherlands

Address for correspondence:

C. (Linda) M.C. van Campen,

Planetenweg 5,

2132 HN Hoofddorp

Netherlands

Tel: +31206597888

Fax: +31205241235

Email address: info@stichtingcardiozorg.nl

Abstract

Introduction: muscle pain/fibromyalgia (FM) is common among individuals
with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We
recently demonstrated that during orthostatic stress testing, adults
with ME/CFS reported increased pain. In the current study, we
hypothesized that pain pressure thresholds (PPT) would decrease and
temporal summation (windup) would increase after head-up tilt testing
(HUT), and that the presence of co-morbid FM would be associated with
greater change in both measures.

Methods: We studied adult ME/CFS patients undergoing HUT. PPT and
temporal summation (or windup) measurements were obtained pre- and
post-HUT at the finger and shoulder.

Results: 248 ME/CFS patients (164 with FM and 84 without FM), and 22
healthy controls (HC) were analyzed. In HC there were no significant
differences in PPT between pre- and post-HUT (finger: from 4.7(1.6) to
4.4(1.5); shoulder: from 2.8(1.0) to 2.9(1.0)). In ME/CFS patients with
and without FM, a significant decrease in PPT post-HUT was found
compared to HC (both p\textless{}0.0001). Patients with FM had a lower
PPT pre- and post-HUT (finger: from 2.0(0.9) to 1.5(0.8); shoulder: from
1.2(0.5) to 1.0(0.5) compared to patients without FM (finger: from
5.0(1.6) to 3.3(1.5); shoulder: from 2.2(0.9) to1.9(1.0) (p ranging from
0.001 to \textless{}0.0001). Windup in HC did not significantly change
from pre- to post-HUT. In ME/CFS patients with and without FM windup was
increased compared to HC pre-HUT (both p\textless{}0.0001), but did not
significantly change post-HUT.

Conclusions: Pressure pain threshold decreased in ME/CFS patients with
or without fibromyalgia after head-up tilt test (HUT), but did not
change post-HUT in healthy controls. Windup pre-and post-HUT was
significantly higher compared to healthy controls, but did not change
from pre- to post-HUT. These results demonstrate that, like exercise,
orthostatic stress can negatively influence the physiology of pain
perception in ME/CFS.

\textbf{Keywords} : pressure pain threshold, temporal summation, windup,
fibromyalgia, orthostatic intolerance, tilt table test, myalgic
encephalomyelitis, chronic fatigue syndrome.

\textbf{Introduction}

Apart from the characteristic fatigue, patients with myalgic
encephalomyelitis/chronic fatigue syndrome (ME/CFS) often experience
chronic widespread and persistent pain(1-3). A population-based study
revealed that 94\% of the persons diagnosed with ME/CFS report muscle
pain, and 84\% report joint pain(4). In fact, there is a great overlap
between ME/CFS and fibromyalgia (FM), a disease particularly
characterized by musculoskeletal pain(1).

As has been demonstrated in those with FM(2, 3), previous studies define
central sensitization as the underlying mechanism maintaining chronic
pain in ME/CFS patients(5-7). Central sensitization comprises
hyper-excitement of the central neurons, altered sensory processing in
the brain and malfunctioning descending pain inhibitory mechanisms(8).
Furthermore, exercise-induced pain inhibition is not activated in
patients with ME/CFS, resulting in lower pain thresholds and pain
exacerbation after exercise(9-11).

There are several methods for estimating the intensity of a stimulus
required to evoke a painful sensation in the skin: mechanical, chemical,
electrical, and thermal(12). Algometry is a widely used technique for
determining the effect of mechanical stimulation. It generates data on
pressure pain thresholds (PPT)(13), or the degree of pressure required
before the individual experiences pain. Lower thresholds indicate that
pain is experienced with less pressure. Algometry is valid and
reproducible, with high levels of interrater reliability(14-17). PPT is
one of the most frequently methods used in FM to objectively assess
severity(18-23).

In ME/CFS patients studies have shown that pain is increased following
exercise stress testing(24, 25). Furthermore, studies have shown that
submaximal physical stress reduced pain thresholds both in FM and non-FM
ME/CFS patients, whereas in healthy controls pain thresholds
increased(11, 26, 27).

Another aspect of pain perception that can be studied using algometry is
temporal summation (windup), which refers to an increased pain
perception in response to repetitive painful stimuli. A meta-analysis in
FM patients showed a significant increase in windup compared to HC(28).

In a previous study in ME/CFS patients, we demonstrated that orthostatic
stress results in a cerebral blood flow reduction, and that the blood
flow reduction is associated with onset or worsening of pain(CNP2019).
In the current study, we hypothesized that PPT would be reduced and
windup would increase after orthostatic stress testing in ME/CFS
patients. For this purpose PPT and windup were measured before and after
a head-up tilt test (HUT) in ME/CFS patients and in healthy controls
(HC). We also hypothesized that among ME/CFS participants, the presence
of co-morbid FM would lead to lower PPT and increased windup compared to
those without FM.

\textbf{Materials and Methods}

\emph{Eligible participants} :

Female individuals diagnosed with ME/CFS, who underwent HUT at Stichting
CardioZorg between November 2014 and April 2018 because of a clinical
suspicion of orthostatic intolerance (OI), and in whom a complete set of
the PPT's pre- and post-HUT was available, were included in this study.
Male patients were not studied because of differences in PPT values
compared to female patients. ME/CFS was considered present if
participants met both the 1994 International Chronic Fatigue Syndrome
Study Group criteria for CFS(29) and the 2011 international consensus
definition of ME(30), taking the exclusion criteria into account. During
the first visit ME/CFS patients were classified as having FM or not. FM
was considered present when the diagnosis had been confirmed in the past
by a rheumatologist or when patients fulfilled the criteria of FM based
on the American College of Rheumatology (ACR) fibromyalgia
questionnaire(31). For comparison 22 healthy female subjects underwent
the same test.

The study was carried out in accordance with the Declaration of
Helsinki. The use of clinical data for descriptive studies (PT1450) and
the use of HC (P1411) was approved by the ethics committee of the
Slotervaart Hospital, the Netherlands. All patients gave informed
consent.

\emph{Head-up tilt table test} :

The HUT was performed as described previously(32). Briefly, testing was
conducted at least 3 hours after a light meal. Participants were
encouraged to ingest an ample amount of fluid on the day of the
procedure, but did not drink fluids in the 2 hours before the test.
Participants were studied in a climate-controlled room where the
temperatures ranged from 22-240 \selectlanguage{english}C. Individuals were studied in the
supine position for 15 minutes, and for 30 minutes in the upright
position (70-degrees). The test was ended after 30 minutes, at the
request of the patient, or if the individual developed syncope or
pre-syncope.

Heart rate (HR), systolic and diastolic blood pressures (SBP and DBP)
were continuously recorded by finger plethysmography using the Nexfin
device (BMeye, Amsterdam, NL)(33, 34). An independent radio-controlled
clock was used to mark the starting time of HR and BP recordings as well
as the time of the start of tilting. HR and BP data were extracted from
the Nexfin device and imported into an Excel spreadsheet. Supine HR and
BP data were calculated from the last minute before tilting. Upright HR
and BP data were calculated from the last minute of the upright
position, and referred to as the end of study (EOS) values. Nasal prongs
were placed to measure expired carbon dioxide (CO\textsubscript{2})
concentrations. For the tilt testing component, individuals being
treated with medication that could alter HR or BP (beta-adrenergic
antagonists, midodrine, fludrocortisone, desmopressin, pyridostigmine
bromide, anti-hypertensive medications, or ivabradine) were excluded
from this analysis. Individuals being treated with selective serotonin
reuptake inhibitors or serotonin norepinephrine reuptake inhibitors
continued to take these medications. Patients using neuropathic pain
medication (opioids, anti-depressants, anti-epileptics, low dose
naltrexone) were also allowed to continue the medication.

\emph{Pressure pain thresholds (PPT) and temporal summation (windup):}

PPT and windup measurements were performed by one experienced clinician
(FCV).Prior to the start of PPT and windup measurements, patients rated
their pain at that moment using a paper with a numeric rating scale
(NRS) with the numbers placed vertically and on which anchors were given
at 0: no pain, at 1: very mild pain, at 4: moderate pain, at 8: very
strong pain, and at 10: the worst imaginable pain possible. PPT was
measured with an analogue Fisher algometer (FDK 40, Wagner Instruments,
Greenwich). For this purpose a rubber tip of 1cm\selectlanguage{ngerman}² was placed on the
dorsal surface of the right hand middle finger midway between the first
and the second digital joint, and at the middle of the trapezius muscle
of the non-dominant shoulder. The rubber tip was placed perpendicular to
the skin surface. The right middle finger was chosen irrespective of the
left or right dominance, because the left hand middle finger was used
for the Nexfin BP measurements. The force was gradually increased at a
rate of 1 kg/s until the subject indicated that the pain level had been
reached. At the two sites the procedure was performed three times with
10 sec intervals. The PPT was calculated as the mean from the last two
PPT measurements.

Windup was elicited by 10 applications of the algometer at previously
determined PPT value on the same locations as used for PPT
measurements(35). Windup measurements started at least 3 minutes after
PPT measurements. For each stimulus, pressure was increased at a rate of
1 kg/s to the previously determined PPT, where it was maintained for 1
second before being released. Stimuli were presented with an interval of
2 seconds. The same NRS paper was used as described above. Subjects were
instructed to rate the pain level ranging from 0 to 10 immediately after
each stimulus.

\emph{Statistical analysis}

Data were analyzed using the statistical package of Graphpad Prism
version 6.05 (Graphpad software, La Jolla, California, USA). All
continuous data were tested for normal distribution using the
D'Agostino-Pearson omnibus normality test, and presented as mean (SD) or
as median with the IQR, where appropriate. Nominal data were compared
using the Chi-square test. Groups were compared using the ANOVA test for
unpaired data. A post-hoc analysis was done where ANOVA showed
significant differences, using the t-test. For normally distributed
data, comparing pre-HUT and post-HUT values, we used paired t test. In
data that were not normally distributed the Mann-Whitney test was used.
Due to the multiple comparisons, we chose a conservative p-value of
\textless{}0.01 to be statistically significant.

\textbf{Results}

A total of 313 individuals with diagnosed ME/CFS underwent HUT during
the study period. PPT testing was not performed due to increased
severity of hand pain (often in those with hypermobile joints) (n=50);
15 patients refused PT testing. None of the patients used HR or BP
altering drugs before the measurements. This left 248 females to be
analyzed. One-hundred-sixty-four patients (66\%) fulfilled the criteria
for FM, eighty-four (34\%) did not. As part of the ME/CFS criteria,
patients were asked for the presence of muscle complaints. In the group
of ME/CFS patients without FM 59/84 (70\%) reported muscle pains. In
ME/CFS patients with FM, all patients 164/164 (100\%) reported muscle
pains.

Table 1 shows the demographic characteristics of the study population.
The NRS pain score was significantly different between the three groups
(p all \textless{}0.0001). ME/CFS patients with and without FM showed
higher supine heart rates compared to HC (p\textless{}0.0005 and
p\textless{}0.002, respectively) and higher EOS heart rates compared to
HC (both p\textless{}0.0001). No other variables were significantly
different.

Table 2 shows PPT pre- and post-HUT for the finger and the shoulder. PPT
of HC were all significantly higher than of ME/CFS patients (p ranging
between \textless{}0.005 and \textless{}0.0001), except for PPT of the
finger in ME/CFS patients without FM (p=0.41). Ninety-six ME/CFS
patients (38\%) used neuropathic pain medication, 14/84 (17\%) in
patients without FM and 82/164 (50\%) in patients with FM. A subgroup
analysis showed no differences in PPT between ME/CFS patients with FM
using neuropathic pain medication compared to those without. Similarly,
no differences in PPT were found between ME/CFS patients without FM
using neuropathic pain medication or not (data not shown). Figure 1
shows PPT pre- and post-HUT for the finger and the shoulder in HC (panel
A), in ME/CFS patients without FM (panel B), and in ME/CFS patients with
FM (panel C). PPT of the finger were significantly higher than PPT of
the shoulder in all 3 groups (all p\textless{}0.0001). PPT of HC did not
differ pre- and post-HUT for both the finger and the shoulder (p=0.14
and p=0.54, respectively). In both ME/CFS patient groups there was a
significant difference pre- and post-HUT for both the finger and the
shoulder (p ranging between 0.0001 and \textless{}0.0001). PPT of ME/CFS
patients with FM were significantly lower than PPT of ME/CFS patients
without FM (all p\textless{}0.0001).

Figure 2 shows the graphical representation of the regression line in
the 3 groups for the finger (panel A) and the shoulder (panel B). For
all time points and groups, the linear regression of windup from
stimulus one to ten on both finger and shoulder were highly significant
(p ranging from 0.0003 to \textless{}0.0001). The slopes of the
regression lines pre- and post-HUT for both ME/CFS patient groups were
significantly higher than the slopes of the pre- and post-HUT
regressions lines of HC (all p\textless{}0.0001). There was no
difference in the regression line slopes between pre- and post-HUT
within the 3 groups for both the finger and the shoulder. For comparison
with the published literature, stimulus 1 pain values were subtracted
from stimulus 10 pain values to create a delta windup value. As shown in
Figure 3, for both the finger and the shoulder, delta windup values did
not differ between pre- and post-HUT in HC and the 2 ME/CFS patient
groups. For both locations a statistically significant difference was
found between HC and the 2 ME/CFS patient groups (both
p\textless{}0.0001).

\textbf{Discussion}

In ME/CFS patients sub grouped by the presence or absence of
fibromyalgia (FM), we studied pressure pain thresholds (PPT) and the
effects of orthostatic stress testing on PPT. First, in the supine
position, PPT of the shoulder of ME/CFS patients with and without FM
were all significantly lower than of the pre-HUT PPT of HC. The pre-HUT
PPT of the finger of ME/CFS patients without FM were not different from
that of HC, while that of patients with FM were lower that of HC.
Second, post-HUT PPT declined significantly in ME/CFS patients with and
without FM, whereas it did not change in HC. Third, the temporal
summation (windup), defined by the slopes of the pain severity versus
the number of stimuli and also defined by the difference in pain
sensation between the first and tenth stimulus, were all higher in
ME/CFS patients than in HC (all p\textless{}0.0001). Comparing pre- and
post-HUT slopes, there were no significant differences in both the two
patient groups and in HC, nor was there a difference in pre- and
post-HUT windup defined by the delta pain sensation of the first and
tenth stimulus.

Baseline PPT: the PPT pre-HUT findings in our study of 164 ME/CFS
patients with FM confirm and extend previous reports showing that FM
patients have lower PPT values than HC(18, 36-38). In two ME/CFS studies
a lower baseline PPT was found compared to HC(26, 39). ME/CFS patients
with FM had a lower PPT than ME/CFS patients without FM(40).

In our study the ME/CFS patients without FM had a similar baseline PPT
on the finger compared to HC, and a lower baseline PPT on the shoulder
compared to HC. A recent study in HC by Park et al. showed higher PPT on
hands and fingers compared to the PPT of muscular parts, indicating the
finger to be relatively less sensitive to pain(41). Despite the absence
of a difference in PPT of the finger in patients without FM, PPT of the
shoulder were significantly lower than that of HC. This could be
explained by a high prevalence of muscle pain in even in patients
without FM: in our study 70\% of the ME/CFS patients without FM reported
muscle pain and 17\% of patients without FM used neuropathic pain
medication. Also, NRS pain scores of patients without FM were higher
than the NRS pain scores of HC. Taken together, our data on PPT and NRS
pain scores and the data of Geisser et al.(40) indicate that pain is a
very common phenomenon in adults with ME/CFS, with the pain spectrum
ranging from no pain to severe pain/fibromyalgia. Therefore, not only
the presence or absence of FM should be taken into account in pain
management, but also the PPT values of the patients, irrespective of the
diagnose of FM. This approach of using PPT measurements warrants further
study.

PPT post-stressor: Earlier studies of the response of PPT to a
physiologic stressor among HC have primarily used exercise as the
intervention. Studies in HC invariably show that PPT is higher after
exercise, indicating hypoalgesia (see for a review Koltyn)(42). In FM
patients the data on PPT post-exercise are conflicting: in 2 studies an
unchanged PPT in the non-exercised muscle groups were found after
isometric contraction exercise(36, 43). In contrast, in two other
studies an increased PT in the non-exercised muscle group was found in
FM patients(37, 38). In ME/CFS patients a different pattern was seen:
post-exercise PPT increased in HC in contrast to a decrease in ME/CFS
patients(11, 26). In the present study, PPT in HC after the orthostatic
stressor were unchanged, whereas in both ME/CFS patient groups PPT were
significantly lower compared to values pre-HUT (both
p\textless{}0.0001).

In a recent study we showed that during HUT, adults with ME/CFS reported
increased fatigue, decreased concentration, increased
dizziness/light-headedness, and the provocation or worsening of
pain(NCP2019). Moreover, those with ME/CFS experienced a significant
decrease of cerebral blood flow compared to HC, and that there was an
inverse linear relation between the number of symptoms reported during
HUT and the reduction in cerebral blood flow. In other studies, it was
shown that working memory function, as assessed by the n-back test,
decreased during HUT(44, 45). We therefore hypothesize, that the pain
perception increase, as demonstrated by a PPT decrease, may be related
to reduction in cerebral blood flow. The pathophysiology of the
increased pain sensation after orthostatic stress (possibly related to
increased catecholamines, metabolic changes, or inflammatory changes)
needs to be addressed in future studies. On the other hand prolonged
standing as a physiologic stressor in ME/CFS patients, might also be
responsible for the increase in PPT. Future interventions during HUT,
like application of a lower body compression could address the question
whether the PPT decrease is due to cerebral blood flow reduction or due
to prolonged standing(46).

Baseline windup: a recent meta-analysis comparing FM patients with HC
showed that windup was significantly higher in FM patients compared to
HC (test for overall effect: p=0.0005)(28). This meta-analysis analyzed
14 studies, including 298 healthy controls and 318 FM patients. In an
ME/CFS study windup in ME/CFS patients (n=48) with a high pain rating
score, a non-significant difference compared to HC (n=39) was found(47).
Our results show, both in ME/CFS patients with and without FM, a highly
significantly increased windup in ME/CFS patients (both
p\textless{}0.0001) compared to HC. The differences between our study
and of Collin et al.(47) are unexplained but may be due to different
inclusion criteria.

Windup post-stressor\textbf{:} in a study using thermal stimulation, a
differential effect of exercise was shown in HC (n=10) vs FM patients
(n=10)(48). Following a maximal exercise stress test in FM patients,
windup was higher than pre-exercise data, whereas in HC windup was lower
post-exercise. In the study of Malfliet et al. post submaximal exercise
windup between 20 HC and 20 ME/CFS patients no significant differences
were observed(49). In the present study pre-HUT windup of HC was
significantly lower than windup of ME/CFS patients with and without FM.
Post-HUT data did not change in HC and in the two patient groups. A
review of Staud et al. without pointing out any number of patients
involved, indicated that part of the windup is related to abnormal pain
processing in the spinal cord(2). Possibly, differences in flow
reduction of the spinal cord vs central cortical and subcortical areas
or different sensitivities to flow reduction may explain the observed
differences in PPT and windup, but needs to be studied further.

\emph{Limitations.}

The reliability of PPT measurements is dependent upon not only the
application technique of the observer, but also on the ability of the
participant to provide a consistent verbal indication of the PPT level.
It has been previously shown in ME/CFS patients that working memory, as
assessed by n-back testing, is reduced during-HUT compared to
pre-HUT(44, 45). This may have altered the post-HUT assessment of PPT
and windup. For reasons of impaired memory and concentration we applied
1 kg/s instead of 2 kg/s in determining windup, as patient responses
might be delayed(35). For the same reason we asked patients to rate
windup after every stimulus. Whether this may change the outcomes, needs
to be studied in future. The reduction of cerebral blood flow during
HUT(50) is variable between ME/CFS patients. To which extent the
cerebral blood flow reduction is related to PPT changes is subject of
another study. We only studied ME/CFS patients undergoing HUT because of
the suspicion of OI. It is unknown whether ME/CFS patients without OI
show the same PPT and windup alterations. Also, the effect in male
ME/CFS patients is unknown. This single center study is the first on the
effects of HUT on PPT and windup and needs to be replicated by others.

\emph{Conclusions.}

This is first study showing the influence of orthostatic stress on pain
perception in ME/CFS patients. After orthostatic stress PPT is decreased
in ME/CFS patients, with or without fibromyalgia, whereas in healthy
controls PPT did not change. In contrast, after orthostatic stress
windup is unchanged in both HC and the two patient groups, but
significantly increased in the two patient groups compared to HC.

\textbf{Figure Legends}

Figure 1. Pressure Pain thresholds of the finger and shoulder pre- and
post-HUT. Panel A shows healthy controls, panel B ME/CFS patients
without fibromyalgia and panel C ME/CFS patients with fibromyalgia. HUT:
head-up tilt test; PPT: pressure pain thresholds.

Figure 2. Temporal summation on the finger and shoulder pre- and
post-HUT for HC, ME/CFS patients without and with fibromyalgia. For
clarity of the figure mean +/- SEM are shown. Panel A shows the windup
for the finger and Panel B for the shoulder. The slopes within the
groups (pre- and post-HUT) were not statistically significant.

FM: fibromyalgia, +: with; -: without; HC: healthy controls; pre: before
head-up tilt test; post: after head-up tilt test

Figure 3. Delta windup (stimulus 10 minus stimulus 1) pre- and post-HUT
in healthy controls, in ME/CFS patients without fibromyalgia and in
ME/CFS patients with fibromyalgia. Panel A shows the windup for the
finger and Panel B for the shoulder.

FM: fibromyalgia; HUT: head-up tilt test

\textbf{Table 1.} \textbf{Demographic data and hemodynamic HUT results
of the study population*}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 1 HC\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 2 ME/CFS without FM\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 3 ME/CFS with FM\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
ANOVA 1 vs 2 vs 3\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 2 vs 3\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 1 vs 2\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 1 vs 3\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Age (years)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
41(14)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
40 (12)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
38 (11)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.28\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Disease duration (years)*\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
10 (4-17)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
9 (6-15)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.84\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Height (cm)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
171 (5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
171 (7)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
170 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.43\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Weight (kg)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
72 (16)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
71 (14)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
72 (16)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.74\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
BMI (kg/m\textsuperscript{2})\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
24.8 (5.1)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
24.2 (5.0)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
25.0 (5.6)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.51\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NRS pain*\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0 (0-1.3)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
3 (1-5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5 (3-6)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Heart rate supine (bpm)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
66 (13)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
79 (8)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
77 (12)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.31\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.002\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0005\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Heart rate EOS (bpm)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
80 (14)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
98 (18)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
103 (20)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.08\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
SBP supine (mmHg)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
137 (16)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
137 (16)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
136 (15)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.84\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
SBP EOS (mmHg)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
125 (16)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
123 (19)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
125 (18)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.81\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
DBP supine (mmHg)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
79 (7)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
79 (8)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
79 (7)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.87\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
DBP EOS (mmHg)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
81 (9)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
82 (12)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
83 (10)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.57\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

DBP: diastolic blood pressure; EOS: end of study; FM: fibromyalgia; HC:
healthy controls; SBP: systolic blood pressure; NRS: numeric rating
scale. *data with median and IQR.

\textbf{Table 2.} \textbf{Pressure pain threshold (PPT) data pre- and
post-HUT of the study population}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 1 HC\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 2 ME/CFS without FM\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 3 ME/CFS with FM\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
ANOVA 1 vs 2 vs 3\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 2 vs 3\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 1 vs 2\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Group 1 vs 3\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PPT finger pre-HUT\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
4.7 (1.6)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5.0 (1.6)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2.0 (0.9)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.41\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PPT finger post-HUT\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
4.4 (1.5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
3.3 (1.5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.5 (0.8)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PPT shoulder pre-HUT\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2.8 (1.0)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2.2 (0.9)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.2 (0.5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.005\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PPT shoulder post-HUT\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2.9 (1.0)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.9 (1.0)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.0 (0.5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

FM: fibromyalgia; HC: healthy controls; HUT: head-up tilt test; PPT:
pressure pain threshold.

Figure 1 A-C Pressure Pain thresholds of the finger and shoulder pre-
and post-HUT.

Figure 2 A-B Temporal summation on the finger and shoulder pre- and
post-HUT for HC, ME/CFS patients without and with fibromyalgia.

Figure 3 A-B Delta windup (stimulus 10 minus stimulus 1) pre- and
post-HUT in healthy controls, in ME/CFS patients without fibromyalgia
and in ME/CFS patients with fibromyalgia

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\textbf{Acknowledgements}

Dr. Rowe is supported by the Sunshine Natural Wellbeing Foundation
Professorship of Chronic Fatigue and Related Disorders.

\section*{Conflict of Interest}

{\label{conflict-of-interest}}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

\section*{Author Contributions}

{\label{author-contributions}}

CMCVC, PCR, and FCV conceived the study, CMCVC and FCV collected the
data, CMCVC performed the primary data analysis and FCV and PCR
performed secondary data analyses. All authors were involved in the
drafting and review of the manuscript.

\textbf{Funding}

This study was performed without grant funding.

\section*{Data Availability Statement}

{\label{data-availability-statement}}

The raw data supporting the conclusions of this manuscript will be made
available by the authors, without undue reservation, to any qualified
researcher.\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Fig1AC-pressure-pain-threshold-hv-and-2-pt-groups/Fig1AC-pressure-pain-threshold-hv-and-2-pt-groups}

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\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Fig-2AB-temperal-summation-hv-and-2-pt-groups/Fig-2AB-temperal-summation-hv-and-2-pt-groups}

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\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Fig-3AB-temperal-summation-10-1-hv-and-pts/Fig-3AB-temperal-summation-10-1-hv-and-pts}

\end{center}
\end{figure}

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