Abstract

   Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome
   (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is
   poorly defined, the pathophysiology is unknown, and no
   disease-modifying treatments are available. We used rigorous criteria
   to recruit PI-ME/CFS participants with matched controls to conduct deep
   phenotyping. Among the many physical and cognitive complaints, one
   defining feature of PI-ME/CFS was an alteration of effort preference,
   rather than physical or central fatigue, due to dysfunction of
   integrative brain regions potentially associated with central catechol
   pathway dysregulation, with consequences on autonomic functioning and
   physical conditioning. Immune profiling suggested chronic antigenic
   stimulation with increase in naïve and decrease in switched memory
   B-cells. Alterations in gene expression profiles of peripheral blood
   mononuclear cells and metabolic pathways were consistent with cellular
   phenotypic studies and demonstrated differences according to sex.
   Together these clinical abnormalities and biomarker differences provide
   unique insight into the underlying pathophysiology of PI-ME/CFS, which
   may guide future intervention.

   Subject terms: Neurological disorders, Infectious diseases, Diseases of
   the nervous system
     __________________________________________________________________

   Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome
   (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is
   poorly defined and the pathophysiology unknown. Here, the authors
   conduct deep phenotyping of a cohort of PI-ME/CFS patients.

Introduction

   Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is the
   commonly used term to describe a disorder of persistent and disabling
   fatigue, exercise intolerance, malaise, cognitive complaints, and
   physical symptoms with significant socioeconomic consequences. The
   physiological mechanism responsible for the persistence of fatigue and
   related symptoms has yet to be determined. Immunologic^[162]1,
   bioenergetic^[163]2–[164]5, and physiologic^[165]6 alterations have
   been reported. However, many of these findings have been inconsistent
   and both their clinical relevance and relation to each other are
   unclear. Hence, there are currently no effective disease modifying
   treatments for ME/CFS, and even developing and testing of potential new
   treatments is hampered by difficulty in defining cases or tracking
   response through symptoms or biomarkers.

   A major obstacle to rigorous ME/CFS research is case assessment due to
   the absence of a diagnostic biomarker. Over 20 different diagnostic
   criteria underscore the difficulty in defining the clinical symptom
   content of ME/CFS^[166]7. The usual symptoms are non-specific and
   overlap with other diseases, hence misattribution is frequent and
   ME/CFS is typically a diagnosis of exclusion^[167]8. ME/CFS often
   occurs following an acute infection, designated post-infectious-ME/CFS
   (PI-ME/CFS), with an estimated incidence of 10–12% after certain
   infections^[168]9,[169]10.The most well-known association of PI-ME/CFS
   has been with the Epstein-Barr Virus^[170]11 but as the full extent of
   the sequelae of the COVID-19 pandemic are better understood, SARS-CoV-2
   may become an even stronger correlate^[171]12.

   In 2016, the National Institutes of Health (NIH) launched an initiative
   to study ME/CFS. The NIH Division of Intramural Research developed an
   exploratory clinical research program to perform deep phenotyping on a
   cohort of PI-ME/CFS participants and healthy volunteers (HV) as
   controls. Prior to the SARS-CoV-2 pandemic, this study recruited a
   cohort of well-characterized PI-ME/CFS patients and applied modern
   broad and deep scientific measures to describe their biophenotype
   compared to HVs. The aim was to identify relevant group differences
   that could generate new hypotheses about the pathogenesis of PI-ME/CFS
   and provide direction for future research. Over 75 scientists and
   clinicians across 15 of the 27 institutes that comprise the NIH
   contributed to this multi-disciplinary work. Importantly, we developed
   rigorous inclusion criteria which comprised detailed medical and
   psychological evaluations to minimize diagnostic misattribution. A
   relatively homogenous population was recruited in whom symptoms were
   initiated after infection. This study aimed to investigate the
   underlying pathophysiological mechanisms. The participants underwent a
   multi-dimensional evaluation that included a wide range of
   physiological measures, physical and cognitive performance testing, and
   biochemical, microbiological, and immunological assays of blood,
   cerebrospinal fluid, muscle, and stool. Measurement techniques were
   developed to query issues such as physical capacity, effort preference,
   and deconditioning that may confound the results. Multi-omic
   measurements of gene expression, proteins, metabolites, and lipids were
   performed in parallel on collected samples. This report summarizes and
   contextualizes the main findings arising from this work.

Results

   Data are reported in the following formats: (HV mean ± standard
   deviation versus PI-ME/CFS mean ± standard deviation, p-value). Odds
   ratio (OR) and relative odds ratio (ROR) are reported as: HV:PI-ME/CFS
   ratio [95% confidence interval]. Additional analysis and a glossary of
   abbreviations are provided in the Supplementary Information.

Case ascertainment

   Study recruitment occurred between December 2016 and February 2020
   (Fig. [172]1a). Of 484 ME/CFS inquiries, 217 individuals underwent
   detailed case reviews entailing telephone interviews and medical record
   review (Supplementary Data S[173]2). Of these, 27 underwent in-person
   research evaluation and 17 were determined to have PI-ME/CFS by a panel
   of clinical experts with unanimous consensus. Twenty-one comparator
   healthy volunteers were recruited separately. Additional recruitment
   was terminated due to the COVID-19 pandemic.

Fig. 1. Patient recruitment and characteristics.

   [174]Fig. 1
   [175]Open in a new tab

   a Diagram showing the procedure followed to recruit adjudicated
   Post-infectious Myalgic Encephalomyelitis/Chronic Fatigue Syndrome
   (PI-ME/CFS) and matched healthy volunteers (HV) and measurements
   undertaken for deep phenotyping of the cohorts. The number of
   participants at each stage of recruitment are noted. b Comparisons of
   age, sex, and BMI distribution in HV (blue; n = 21 independent
   participants) and PI-ME/CFS (red; n = 17 independent participants)
   using unadjusted two-sided t-tests for independent samples. c
   Distribution of the response of HV (blue; n = 21 independent
   participants) and PI-ME/CFS (red; n = 17 independent participants) to
   the indicated patient reported outcome questionnaires. Group
   comparisons performed using unadjusted two-sided Mann–Whitney-U tests.
   CSF cerebrospinal fluid, PBMC peripheral blood mononuclear cell, BMI
   body mass index, PROMIS Patient-Reported Outcomes Measurement
   Information System, PHQ-15 Patient Health Questionnaire – 15, MASQ
   Multiple Ability Self-Report Questionnaire. Figure 1A created with
   Biorender.com. Source data are provided as a Source Data file.

Cohort characteristics

   Characteristics of the participants are detailed in Fig. [176]1b, c and
   Supplementary Data S[177]5; there were no group differences in age,
   sex, or BMI. There were no group differences in performance validity
   testing (Supplementary Data S[178]6); a method for determining whether
   neuropsychological test performances are overly impacted by
   non-cognitive factors^[179]13. Patient reported outcome measurement
   information system (PROMIS) and other questionnaires for fatigue,
   emotional distress, sleep disturbances, anxiety and other symptoms were
   greater (p < 0.05) in PI-ME/CFS than HVs (Fig. [180]1c, Supplementary
   Data S[181]7). There were no clinically relevant findings on physical
   examination, psychological evaluation, or laboratory testing in either
   group (Supplementary Data S[182]8, [183]9). Measurements of small nerve
   fiber density and neuronal injury markers in blood and cerebrospinal
   fluid were not different between groups (Supplementary Fig. [184]S1).
   Polysomnography did not reveal clinically relevant findings
   (Supplementary Information, Sleep dysfunction). The groups did not
   differ in composition by whole-body dual energy X-ray absorptiometry or
   in slow-to-fast muscle fiber atrophy^[185]14 as measured by Type 2:
   Type 1 muscle fiber median Feret diameter ratio (Type2:1 mFd) using
   ATPase pH 9.4 stain of the vastus lateralis (Supplementary
   Figs. [186]S2 and [187]S3D).

Autonomic dysfunction as measured by multiple parameters in PI-ME/CFS

   Head-up tilt table testing for up to 40 min showed no group differences
   in frequency of orthostatic hypotension, excessive orthostatic
   tachycardia, or tilt-related symptoms requiring test cessation.
   Twenty-four hour ambulatory ECG showed that PI-ME/CFS participants had
   diminished heart rate variability (HRV) in SDNNi, rMSSD, and pNN50 time
   domain indices (Fig. [188]2a–d). PI-ME/CFS participants also had
   altered frequency domain differences, with decreased high frequency
   (HF) and low frequency (LF) power (Fig. [189]2e, f). Non-linear
   analyses adjusted by hour of the day further demonstrated group
   differences in (p = 1.1E−12), SD2 (p = 2.1E−05), and SD1/SD2
   (p = 3.9E−07). Similarly, group heart rates displayed two notable
   trends (Fig. [190]2g). Increased heart rate in PI-ME/CFS participants
   throughout the course of a day suggests comparatively increased
   sympathetic activity. PI-ME/CFS participants also had a diminished drop
   in nighttime heart rate suggesting diminished parasympathetic activity,
   consistent with observed differences in rMSSD, HF power, pNN50, and
   SD1. A decrease in baroslope (Fig. [191]2h) and longer blood pressure
   recovery times after the Valsalva maneuver (3.0 ± 0.2 versus
   4.1 ± 0.4 sec, p = 0.014) in PI-ME/CFS reflect decreased
   baroreflex-cardiovagal function. Considered together, these data
   suggest that there is an alteration in autonomic tone, implying central
   nervous system regulatory change.

Fig. 2. Diminished heart rate variability measures are consistent with
decreased parasympathetic activity in the PI-ME/CFS cohort compared to HV.

   [192]Fig. 2
   [193]Open in a new tab

   a Table of time and frequency domain heart rate variability
   measurements. Group comparisons for panel a were performed with
   unadjusted two-sided Mann–Whitney U tests. Box plots comparing HV
   (blue; n = 19 independent participants) and PI-ME/CFS (red; n = 14
   independent participants) for (b) SDNNI (msec) (c) rMSSD (msec,
   p = 0.019, unadjusted two-sided t-test for independent samples with
   equal variance) (d) pNN50 (%, p = 0.017, unadjusted two-sided
   Mann–Whitney U test) (e) lnHF(ms^2) (f) lnLF (ms^2). Box plots depict
   the median (horizontal line) within quartiles 1–3 (bounds of box).
   Whiskers extend to minimum and maximum values g: Mean heart rate of HV
   (blue; n = 20 independent participants) and PI-ME/CFS (red; n = 13
   independent participants) of 5-min segmented intervals over a 24-h
   period graphed over 24-h period. Error bars represent ±SE for each
   5-min time block for each group. Note HV graph (blue) demonstrates
   fluctuations throughout the day with subject heart rates displaced
   slightly higher, suggesting increased sympathetic activity. Similarly,
   the typical sinusoidal drop in heart rate over sleeping hours is
   diminished in subjects also suggesting diminished parasympathetic
   and/or increased sympathetic activity. h Box plot of
   baroreflex-cardiovagal gain as measured by mean baroslope (ms/mmHg).
   HVs (blue; n = 19 independent participants) and PI-ME/CFS (red; n = 16
   independent participants) are compared using an unadjusted two-sided
   t-test for independent samples with equal variance (p = 0.015). Box
   plot H depicts the median (horizontal line) within quartiles 1–3
   (bounds of box). Whiskers extend to minimum and maximum values. SDNNi
   standard deviation of the average NN intervals for each 5 min segment
   of a 24 h HRV recording, rMSSD root mean square of successive
   differences between normal heartbeats, pNN50 proportion of NN50 divided
   by the total number of NN (R-R) intervals, HF high frequency, LF low
   frequency, SD1 standard deviation of Poincaré plot of RR intervals
   perpendicular to the line-of-identity, SD2 standard deviation of the
   Poincaré plot of RR intervals along the line-of-identity. Source data
   are provided as a Source Data file.

Altered effort preference in PI-ME/CFS

   The Effort-Expenditure for Rewards Task (EEfRT)^[194]15 was used to
   assess effort, task-related fatigue, and reward sensitivity
   (Supplementary Fig. [195]S5A–D). Given equal levels and probabilities
   of reward, HVs chose more hard tasks than PI-ME/CFS participants (Odds
   Ratio (OR) = 1.65 [1.03, 2.65], p = 0.04; Fig. [196]3a). Two-way
   interactions showed no group differences in responses to task-related
   fatigue (Relative Odds Ratio (ROR) = 1.01 [0.99, 1.03], p = 0.53;
   Fig. [197]3a), reward value (ROR = 1.57 [0.97, 2.53], p = 0.07), reward
   probability (ROR = 0.50 [0.09, 2.77], p = 0.43), and expected value
   (ROR = 0.98 [0.54, 1.79], p = 0.95). Effort preference, the decision to
   avoid the harder task when decision-making is unsupervised and reward
   values and probabilities of receiving a reward are standardized, was
   estimated using the Proportion of Hard-Task Choices (PHTC) metric. This
   metric summarizes performance across the entire task and reflects the
   significantly lower rate of hard-trial selection in PI-ME/CFS
   participants (Fig. [198]3a). There was no group difference in the
   probability of completing easy tasks but there was a decline in
   button-pressing speed over time noted for the PI-ME/CFS participants
   (Slope = −0.008, SE = 0.002, p = 0.003; Fig. [199]3b). This pattern
   suggests the PI-ME/CFS participants were pacing to limit exertion and
   associated feelings of discomfort^[200]16. HVs were more likely to
   complete hard tasks (OR = 27.23 [6.33, 117.14], p < 0.0001) but there
   was no difference in the decline in button-press rate over time for
   either group for hard tasks (Fig. [201]3b).

Fig. 3. Impaired effort measures and motor performance were observed in
PI-ME/CFS cohort compared to HV.

   [202]Fig. 3
   [203]Open in a new tab

   a, b Effort-Expenditure for Rewards Task: a Probability of choosing the
   hard task is significantly more in HV (blue, n = 16 independent
   participants) compared with PI-ME/CFS (red; n = 15 independent
   participants) at the start of and throughout the task. The Odds Ratio
   for the probability of choosing the hard task at the start of the task
   is 1.65 [1.03, 2.65], p = 0.04 using Fisher’s Exact test. The lines are
   the curvilinear fits and the error bands are the confidence intervals.
   Decline rates (i.e. response to fatigue) between the groups is similar
   as the trial progresses. b Button press rates for easy (right) and hard
   (left) tasks as the trial progresses is shown for HV (blue) and
   PI-ME/CFS (red) participants. The lines are the linear fits and the
   shaded error bands are the confidence intervals. The decline in button
   press rate over time during the easy tasks in PI-ME/CFS did not impact
   easy task performance, which is supportive of pacing in PI-ME/CFS
   participants. c–e Grip Strength test: Box plots of (c) maximum grip
   force of HV (blue; n = 20 independent participants) and PI-ME/CFS (red;
   n = 16 independent participants) and (d) time to failure of HV (blue;
   n = 18 independent participants) and PI-ME/CFS (red; n = 16 independent
   participants), unadjusted two-sided t-test for independent samples with
   equal variance, p = 0.0002. Correlation between time to failure and (e)
   proportion of hard task choices in HV (blue; n = 15 independent
   participants) and PI-ME/CFS (red; n = 14 independent participants). For
   figure e, the relationship between indicated variables in x and y axis
   were fitted by linear regression in each group. Linear regression
   t-tests were used to determine non-zero slope. Exact p values of the
   correlations are presented on the graph. For box plots c and d, boxes
   depict the median (horizontal line) within quartiles 1–3 (bounds of
   box). Whiskers extend to minimum and maximum values. Source data are
   provided as a Source Data file.

Equivalent motor performance in PI-ME/CFS and healthy volunteers

   On single grip task, maximal voluntary contraction (MVC) was not
   different between groups (Fig. [204]3c) and correlated with lean arm
   mass but not effort preference or Type2:1 mFd (Supplementary
   Fig. [205]S6A–C). Time to failure, the inability to maintain grip force
   at 50% of maximum contraction, was significantly shorter (p = 0.0002)
   in the PI-ME/CFS participants (Fig. [206]3d) and correlated with effort
   preference (Fig. [207]3e) in PI-ME/CFS but not in HVs. Time to failure
   did not correlate with lean arm mass or Type2:1 mFd in either group.

   Repetitive grip testing was performed on a subgroup of participants.
   MVC did not differ between groups. A rapid decline in force along with
   a significantly lower number of non-fatigued blocks (Fig. [208]4a) and
   a relative decrease in the slope of the Dimitrov index^[209]17,[210]18
   (Fig. [211]4b) occurred in PI-ME/CFS participants but both remained
   constant in HVs, suggesting that the decline of force was not due to
   peripheral fatigue or a neuromuscular disorder. Motor Evoked Potential
   amplitudes using transcranial magnetic stimulation of HVs decreased
   over the course of the task, consistent with post-exercise depression
   as seen in healthy and depressed volunteers^[212]19, while they
   increased in PI-ME/CFS participants (Fig. [213]4c). This indicates that
   the primary motor cortex remained excitable for PI-ME/CFS, suggesting
   reduced motor engagement from this group^[214]20.

Fig. 4. Impaired sustained effort and motor performance was observed in
PI-ME/CFS cohort compared to HV.

   [215]Fig. 4
   [216]Open in a new tab

   a–e Repetitive Grip Strength test: a Grip force normalized to maximum
   voluntary contraction (MVC) in the first block, the last block prior to
   fatigue onset, and the first three blocks after fatigue onset in HV
   (blue) and PI-ME/CFS cohorts (red). A significant grip force difference
   was noted between the groups (−1.2 ± 4 versus −6.4 ± 4 kilogram-force
   units, t(12) = 2.46, p = 0.03). The number of non-fatigued grip test
   blocks of HV (blue) and PI-ME/CFS (red) participants is also displayed.
   b Slope of the Dimitrov index across the first block (b[1]), the last
   block prior to fatigue onset (b[n]), and the first three blocks after
   fatigue onset (f[1], f[2], and f[3]) in HV (blue; n = 6 independent
   participants) and PI-ME/CFS (red; n = 8 independent participants)
   patients. A significant difference was noted between the groups
   (0.2 ± 0.5 versus −0.43 ± 0.3, t(12) = 3.2, p = 0.008). c Mean and
   standard error of the motor evoked potential of HV (blue; n = 6
   independent participants) and PI-ME/CFS (red; n = 8 independent
   participants) participants spanning the last five grip test blocks
   prior to fatigue onset. The amplitudes of the MEPs of HVs significantly
   decreased over the course of the task while the amplitudes of the MEPs
   of PI-ME/CFS participants significantly increased (−0.13 ± 0.2 versus
   0.13 ± 0.2 MEP units; t(12) = 2.4, p = 0.03 D. Brain regions where
   Blood Oxygen Dependent (BOLD) signal decreased over grip strength
   blocks in PI-ME/CFS patients and increased over grip strength blocks in
   HVs. e Brain activation of the regions depicted in d measured in the
   blocks of four over the course of the experiment in HV (blue; n = 10
   independent participants) and PI-ME/CFS (red; n = 8 independent
   participants) cohorts. For e, a two-way ANOVA was run where
   F(3,45) = 5.4 with a voxel threshold of p ≤ 0.01, corrected for
   multiple comparisons p ≤ 0.05, k > 65 (p-values were 0.976, 0.43, 0.02
   (*), and 0.02 (*) for blocks 1 to 4 respectively). Source data are
   provided as a Source Data file.

Decreased activation of integrative brain regions in PI/ME-CFS

   Repetitive grip testing by functional brain imaging was performed on a
   subgroup of participants and also showed a rapid decline in force
   (Supplementary Fig. [217]S7E) and a significantly lower number of
   non-fatigued blocks (p = 0.004) in PI-ME/CFS. First, we assessed
   commonly activated brain areas by implementing a conjunction analysis
   in which we took each group t-test across all blocks, thresholded
   voxels at p ≤ 0.01 with a multiple comparison correction at p = 0.05,
   k > 65 and kept voxels that were commonly activated in each group. HV
   and PI-ME/CFS participants showed force-related brain activation in the
   left M1, right cerebellum, and left putamen during the task. We next
   assessed group differences with t-test (at p = 0.01, k > 65), but there
   was no difference between the groups. We also assessed changes across
   blocks with a two-way ANOVA (2 groups × 4 blocks), which showed that
   blood oxygen level dependent (BOLD) signal of PI-ME/CFS participants
   decreased across blocks bilaterally in temporo-parietal junction (TPJ)
   and superior parietal lobule, and right temporal gyrus in
   contradistinction to the increase observed in HVs (F (3,45) = 5.4,
   voxel threshold p ≤ 0.01, corrected for multiple comparisons p ≤ 0.05,
   k > 65; Fig. [218]4d, e). TPJ activity is inversely correlated with the
   match between willed action and the produced movement.

Differential cardiorespiratory performance in PI-ME/CFS

   Cardiopulmonary exercise testing (CPET) was performed on eight
   PI-ME/CFS and nine HVs (Supplementary Fig. [219]S8A). During testing,
   all but one PI-ME/CFS participant reached the peak respiratory exchange
   ratio (RER) of ≥1.1 and there was no difference between the groups.
   Effort preference did not correlate with peak power in PI-ME/CFS
   participants (Supplementary Fig. [220]S8B). The ratio of
   Ventilation/VCO[2], oxygen saturation levels in the quadricep muscle as
   measured by Near Infrared Spectroscopy, and gross mechanical efficiency
   were not different between groups (Supplementary Fig. [221]S8C–E).
   These results suggest that PI-ME/CFS participants performed to the best
   of their abilities.

   Peak power (p = 0.08), peak respiratory rate, peak heart rate
   (p = 0.07), and peak VO2 (p = 0.004) were all lower in the PI-ME/CFS
   participants (Fig. [222]5a–d), a difference of approximately 3.3
   metabolic equivalent of task units (METs). These peak measures did not
   correlate with effort preference in PI-ME/CFS. PI-ME/CFS participants
   performed at a lower percentage of their predicted VO[2peak]
   (Fig. [223]5e) as determined by the Wasserman-Hansen cycling
   equation^[224]21,[225]22 and at a lower percentage of their
   age-predicted maximal heart rate (Supplementary Fig. [226]S8F). The
   lower peak heart rate and higher resting heart rate for PI-ME/CFS
   participants led to a lower heart rate reserve (Fig. [227]5f).
   Chronotropic incompetence, as measured by age-predicted HRR (%pHRR),
   was noted in five of eight PI-ME/CFS and one of nine HVs (X^2 (1,
   n = 17) = 4.9, p = 0.03). The slope of the heart rate response during
   the CPET was also lower in PI-ME/CFS participants compared to HVs
   (1.03 ± 0.16 versus 0.70 ± 0.27, p < 0.01; Fig. [228]5g). Anaerobic
   threshold (AT) was achieved within a similar time period and occurred
   at lower VO2 levels in PI-ME/CFS (Fig. [229]5h), a difference of
   approximately 1.4 METs. VO[2] at AT also correlated with Type2:1 mFd
   (Supplementary Fig. [230]S8J) in PI-ME/CFS but not HVs, evidence of
   concomitant muscular deconditioning. Salivary cortisol measurements
   taken at rest in the morning, at noon, and before sleep showed no group
   differences but were significantly lower in PI-ME/CFS participants one
   hour after CPET (Supplementary Fig. [231]S8L). Thus, despite successful
   CPET engagement, PI-ME/CFS participants were less likely to achieve
   their predicted maximal output, suggesting a differential
   cardiorespiratory performance related to autonomic function,
   hypothalamic-pituitary-adrenal axis hyporesponsiveness, and muscular
   deconditioning from disuse that clinically impacts activities of daily
   life ^[232]23. This was supported by at-home waist actigraphy measures,
   demonstrating a lower step count and total activity for PI-ME/CFS
   participants due to less moderate intensity activity (40.64 ± 37.4
   versus 6.4 ± 7.0 min/day; p = 0.007).

Fig. 5. Impaired cardiopulmonary performance was observed in PI-ME/CFS cohort
compared to HV.

   [233]Fig. 5
   [234]Open in a new tab

   Cardiopulmonary Exercise Test (CPET) a–h: Box plots of (a) peak power
   attained during CPET for HV (blue; n = 9 independent participants) and
   PI-ME/CFS (red; n = 8 independent participants) participants, (b) peak
   respiratory rate with the error bars representing ±SD at each work
   rate, (c) peak heart rate (unadjusted two-sided t-test for independent
   samples with unequal variance, p = 0.072), (d) Peak VO2 (unadjusted
   two-sided t-test with equal variance, p = 0.002), (e) Percent of
   predicted VO2 achieved of HV (blue; n = 9 independent participants) and
   PI-ME/CFS (red; n = 8 independent participants) participants
   (unadjusted two-sided t-test for independent samples with equal
   variance, p = 0.004), and (f) heart rate reserve for HV (blue; n = 9
   independent participants) and PI-ME/CFS (red; n = 8 independent
   participants) participants (unadjusted two-sided t-test for independent
   samples with equal variance, p = 0.011). g Heart rate as a function of
   % total CPET time depicted as expected for age and gender of HV (n = 9
   independent participants) and PI-ME/CFS (n = 8 independent
   participants) (gray solid and dashed lines, respectively, unadjusted
   two-sided Mann–Whitney U test for independent samples). Mean heart rate
   responses from the CPET are depicted for HV (blue line) and PI-ME/CFS
   (red line). A significant difference was observed for the heart rate
   slope between PI-ME/CFS and expected (0.70 ± 0.27 versus 1.05 ± 0.12,
   p = 0.014), but not for HV and expected (1.03 ± 0.16 versus
   1.08 ± 0.13; p = 0.479). The deviation from expected relation reflects
   chronotropic incompetence in the PI-ME/CFS group. h Box plot of VO2 at
   the anaerobic threshold (AT) in HV (blue; n = 9 independent
   participants) and PI-ME/CFS (red; n = 8 independent participants) using
   an unadjusted two-sided t-test for independent samples with equal
   variance (p = 0.024). For box plots a, c–f, and h boxes depict the
   median (horizontal line) within quartiles 1–3 (bounds of box). Whiskers
   extend to minimum and maximum values. Source data are provided as a
   Source Data file.

   Despite altered cardiorespiratory function, there were no clinically
   meaningful differences in dietary energy intake (Supplementary Data
   S[235]11) or total body energy use, sleeping energy use, or respiratory
   quotient between the groups before or after CPET testing (Supplementary
   Fig. [236]S9A–D; Supplementary Data S[237]12).

Increased cognitive symptoms but normal neurocognitive testing in PI-ME/CFS

   PI-ME/CFS participants had more self-reported total cognitive
   complaints (66.9 ± 14.7 versus 92.1 ± 17.6, p = 0.00006) and in all
   five cognitive domains measured: attention, verbal memory,
   visuoperceptual, language, and visual memory (Fig. [238]1c). In
   contrast, there were no group differences in performance of any of the
   15 neuropsychological tests administered (Supplementary Data S[239]13)
   or differential degradation of performance over time (Supplementary
   Information, Cognitive Function). No correlations were noted between
   any of the 15 neuropsychological tests administered and effort
   preference.

Differential cerebrospinal fluid catechols and metabolite profile in
PI-ME/CFS

   In cerebrospinal fluid, the PI-ME/CFS group had statistically
   significant decreased levels of DOPA, DOPAC, and DHPG (Fig. [240]6a–c).
   Levels of norepinephrine, cys-DOPA, and dopamine did not differ between
   the groups. These results did not change after excluding data from
   participants taking central-acting medications.

Fig. 6. Differential catecholamines and tryptophan pathway metabolites levels
in PI-ME/CFS patients cerebrospinal fluid.

   [241]Fig. 6
   [242]Open in a new tab

   a–c Box plot of the indicated neurotransmitters on y axis in HV (blue;
   n = 21 independent participants) and PI-ME/CFS (red; n = 16 independent
   participants) in (a) unadjusted two-sided Mann–Whitney U test
   (p = 0.021), (b), unadjusted two-sided Mann–Whitney U test (p = 0.025),
   and (c) unadjusted two-sided t-test for independent samples with equal
   variance (p = 0.006). For box plots a–c boxes depict the median
   (horizontal line) within quartiles 1–3 (bounds of box). Whiskers extend
   to minimum and maximum values. Correlation between cerebrospinal fluid
   norepinephrine (NE) and (d) time to failure on grip strength task in HV
   (blue; n = 18 independent participants) and PI-ME/CFS (red; n = 15
   independent participants) or (e) proportion of hard task choices (i.e.,
   effort preference) in HVs (blue; n = 14 independent participants) and
   PI-ME/CFS (red; n = 14 independent participants). f Correlation between
   cerebrospinal fluid dopamine and time to failure in HVs (blue; n = 17
   independent participants) and PI-ME/CFS (red; n = 14 independent
   participants). g Correlation between cerebrospinal fluid DHPG and
   proportion of hard task choices (i.e., effort preference) in HVs (blue;
   n = 14 independent participants) and PI-ME/CFS (red; n = 14 independent
   participants). For figures d–g, the relationship between indicated
   variables in x and y axis were fitted by linear regression in each
   group. The exact p value of each regression is presented on the graph,
   linear regression t-test for nonzero slope. h PCA computed from all
   metabolites measured from the cerebrospinal fluid samples in the
   indicated groups. i Heatmap of statistically significant (false
   discovery rate adjusted p-value < 0.05) differentially expressed
   metabolites in the indicated groups on x axis and the metabolites
   labeled on y axis. Red: upregulated; Blue: downregulated. Supervised
   clustering of metabolites measured from the cerebrospinal fluid samples
   in (j) male cohorts and (k) female cohorts from PLSDA analysis. DHPG
   (S)−3,5-Dihydroxyphenylglycine, PLSDA Partial least square discriminant
   analysis, PC principal component. Source data are provided as a Source
   Data file.

   Additional analysis revealed that norepinephrine correlated with both
   time to failure and effort preference in PI-ME/CFS participants
   (Fig. [243]6d, e). Dopamine correlated with time to failure
   (Fig. [244]6f) but not with effort preference (Supplementary
   Fig. [245]S11B). Among HVs, DHPG correlated with effort preference
   (Fig. [246]6g) but not with time to failure (Supplementary
   Fig. [247]S11C). Several cognitive symptoms correlated with catechols
   in PI-ME/CFS participants (Supplementary Fig. [248]S12A–I). In
   contradistinction, objective neurocognitive performance showed that
   only the Wisconsin Card Sort Test correlated with DOPA in PI-ME/CFS
   participants (Supplementary Fig. [249]S12J). For HVs, several
   correlations between catechols and neuropsychological tests were
   observed (Supplementary Fig. [250]S12K–O). These correlations suggest
   that perception and behavior, but not cognitive performance, are
   related to catechol levels in PI-ME/CFS participants.

   Metabolomic analysis of cerebrospinal fluid also showed group
   differences (Fig. [251]6h). Tryptophan metabolites were among the top
   15 differentially expressed and statistically significant after
   correction for multiple comparisons (Fig. [252]6i). Decreased
   glutamate, dopamine 3-O-sulfate, butyrate, polyamine, and tricarboxylic
   acid (TCA) pathway metabolites were noted in PI-ME/CFS participants
   (Supplementary Data S[253]14A). Threonine and glutamine were decreased
   in males (Fig. [254]6j, Supplementary Fig. S[255]13D, Supplementary
   Data S[256]14C). Several tryptophan metabolites were decreased in
   females suggesting a decrease in serotonin signaling (Fig. [257]6k,
   Supplementary Fig. S[258]13E, Supplementary Data S[259]14D). This was
   irrespective of NSRI/SSRI use.

Immune activation and sex-based differences in PI-ME/CFS

   There were no group differences in percentage of CD4, CD8, and CD19
   cells in both peripheral blood and cerebrospinal fluid (Supplementary
   Data S[260]15A and Supplementary Fig. S[261]15C). An increase in
   percentage of naïve and decrease in switched memory B-cells in blood
   were observed in PI-ME/CFS participants (Fig. [262]7a, b). Two
   PI-ME/CFS participants had detectable antibody secreting B-cells in
   cerebrospinal fluid; these participants did not have oligoclonal bands
   or autoantibodies. There was no group difference in NK cell frequency,
   but a subset of PI-ME/CFS participants had an elevated percentage of NK
   CD56 bright/dim ratio in cerebrospinal fluid. No group differences were
   noted in NK lytic units, CD16/CD56 ratio, and plasma GDF-15 levels in
   blood. Discovery assays for a small panel of autoantibodies detected
   low levels in one PI-ME/CFS and two HVs.

Fig. 7. Flow cytometry demonstrates distinct perturbation in immune cell
subpopulation in PBMCs.

   [263]Fig. 7
   [264]Open in a new tab

   a Boxplot of the B cell subset naïve (%) in HV (blue; n = 20
   independent participants) and PI-ME/CFS (red; n = 17 independent
   participants) groups, unadjusted two-sided t-test for independent
   samples with equal variance (p = 0.037). b Boxplot of B cell switched
   memory in HV (blue; n = 20 independent participants) and PI-ME/CFS
   (red; n = 16 independent participants) groups, unadjusted two-sided
   t-test for independent samples with equal variance (p = 0.008). c
   Boxplot of the CD8 + T cell subset PD-1 (%), Mann–Whitney U test, exact
   p-value = 0.033. d Boxplot of the CD8 + T cell subset CD226 (%),
   unadjusted two-sided t-test for independent samples with equal variance
   (p = 0.055). The samples used for boxplot (d) were collected at a
   separate time point than the others boxplots; HV (blue; n = 7
   independent participants) and PI-ME/CFS (red; n = 8 independent
   participants) groups. e Boxplot of CD8 + T cell CXCR5 (%), unadjusted
   two-sided t-test for independent samples with equal variance
   (p = 0.014). f Boxplot of CD8 + T cell naïve (%), unadjusted two-sided
   t-test for independent samples with equal variance (p = 0.016). Where
   indicated the plots shows the measurements from female and male
   cohorts. Measurements in PBMC samples are shown in shaded box and
   cerebrospinal fluid samples in open box. For box plots a–f boxes depict
   the median (horizontal line) within quartiles 1–3 (bounds of box).
   Whiskers extend to minimum and maximum values. Source data are provided
   as a Source Data file.

   Markers of T-cell activation, PD-1^+ CD8 T-cells, were elevated in the
   cerebrospinal fluid of PI-ME/CFS participants (Fig. [265]7c). PD-1^+
   CD4 T-cells did not change in blood or cerebrospinal fluid. To expand
   on these observations, other T-cell markers (TIGIT, CD244, and CD226)
   were analyzed in a subset of participants. CD226^+ CD8 T-cells were
   decreased in blood (Fig. [266]7d) of PI-ME/CFS participants with no
   change in expression of CD244 or TIGIT (Supplementary Data S[267]15B,
   D).

   When stratified by sex, PI-ME/CFS males had increased CXCR5 expression
   on CD8 + T-cells in cerebrospinal fluid (Fig. [268]7e). PI-ME/CFS
   females had increased CD8+ naïve T-cells in blood (Fig. [269]7f),
   indicating different subpopulation of immune cells are enriched in the
   male and female PI-ME/CFS groups.

   Analysis of PBMC gene expression for all participants by principal
   component analysis (PCA) revealed no outliers (Fig. [270]8a). 614
   differentially expressed (DE) genes clustered the samples based on
   disease status. However, exploration of PCA plot showed that samples
   clustered based on their sex (Fig. [271]8b). Assessment of interaction
   between sex and disease status was not significant. This suggests sex
   is a potential confounder impacting gene expression. Hence DE analysis
   in male and female cohorts was performed, which identified distinct
   subsets of DE genes, with only 34 (<5%) genes overlapping between them
   (Fig. [272]8c, Supplementary Data S[273]16A, B). PCA of DE genes in
   each sex cohort clustered the samples into distinct HV and PI-ME/CFS
   groups (Fig. [274]8d, f).

Fig. 8. Male and female cohorts have distinct perturbation in immune cell
subpopulation and biological processes in PBMCs.

   [275]Fig. 8
   [276]Open in a new tab

   a, b PCA computed from all gene expression values with indicated
   groups: HV (blue), PI-ME/CFS (red) clusters or males (turquoise) and
   females (orange) highlighted for the indicated PCs. c Venn diagram
   showing common DE genes identified from male and female cohorts (p
   value < 0.05 as filter for DE genes using an unadjusted two-sided
   moderated t-test). DE analysis on the (d, e, h, i) male cohorts and (f,
   g, j, k) female cohorts. d, f PCA plots computed from DE genes shows
   robust clustering of samples based on the PI-ME/CFS status. e, g
   Volcano plots shows log transformed statistically significant
   (unadjusted p-values < 0.05 using an unadjusted two-sided moderated
   t-test) up (red) and down regulated (blue) genes in PI-ME/CFS male and
   female cohorts, respectively. h, j Heatmaps of a subset of T cell
   process genes in males and B cell related processes in females. i, k
   Pathway enrichment plots showing top 15 pathways for which the DE genes
   from male and female cohorts selected for. The top 15 pathways are
   labeled on the y axis and the color of the circle is scaled with
   –log-10 p-value and the size of the pathway circles inside the plot are
   proportional to the number of genes that overlapped with the indicated
   pathway. Fisher’s exact test was used in the ‘clusterProfiler’ R
   package to obtain the log-transformed p-values. Red color nodes:
   upregulated in PI-ME/CFS and blue color nodes: downregulated in
   PI-ME/CFS. DE differentially expressed. Source data are provided as a
   Source Data file.

   887 DE genes in male HV and PI-ME/CFS cohorts enriched (p < 0.05) in
   ubiquitin, IL-10, T-cell, and NF-kB pathways (Fig. [277]8h, i). DE
   genes related to the STAT4-TLR9 protein-protein interactome and were
   upregulated in male PI-ME/CFS participants (Supplementary
   Fig. [278]S14A). 849 DE genes identified in female HV and PI-ME/CFS
   cohorts were enriched in B-cell proliferation processes (Fig. [279]8j,
   k) and DE genes identified in the B-cell interactome were upregulated
   in female PI-ME/CFS participants (Supplementary Fig. [280]S14B).
   Additionally, DE genes were also enriched in cytokine and lymphocyte
   proliferation processes. These data are consistent with expansion of
   naïve B-cells by the STAT4-TLR9 and other B-cell pathways observed in
   PI-ME/CFS by flow cytometry.

   PCA analysis of aptamers measured in serum and cerebrospinal fluid did
   not identify outliers. Univariate analysis of the aptamer datasets did
   not identify any FDR-corrected statistically significant differential
   features between PI-ME/CFS and HV participants (Supplementary Data
   S[281]17A, B). Within each sex separately, exploratory multivariate
   analyses suggested a subset of aptamers were predictive of PI-ME/CFS
   status, which are reported for potential validation (Supplementary
   Fig. [282]S15A–D,  [283]S16A–D; Supplementary Data S[284]17C–F).

   Sex differences in PI-ME/CFS were also assessed in a publicly deposited
   ME/CFS RNASeq dataset ([285]GSE130353) from monocytes (Supplementary
   Fig. [286]S17A–D). Of 638 DE genes in males and 420 DE genes in
   females, only 23 genes were common (Supplementary Fig. [287]S17E).
   These observations support that distinct biological processes are
   perturbed in male and female PI-ME/CFS patients.

Sex-based differences in muscle gene expression profile in PI-ME/CFS

   PCA analysis of muscle gene expression did not identify any outliers
   (Fig. [288]9a). Univariate analysis did not identify any FDR-corrected
   statistically significant differential features (Supplementary Data
   S[289]19A). The PCA identified clustering based on sex (Fig. [290]9b).
   Only 15 DE genes were common in male and female cohorts (Fig. [291]9c),
   consistent with prior observations. In the male PCA of 593 DE genes,
   clustering was observed based on the disease status (Fig. [292]9d, e).
   Genes upregulated in the PI-ME/CFS males were enriched in epigenetic
   changes and processing of RNA and the downregulated genes were enriched
   in hexose metabolism and mitochondrial processes (Fig. [293]9h–i).
   Several genes involved in fatty acid beta-oxidation were upregulated,
   in an interactive network (Supplementary Fig. [294]S18A). In the
   females, PCA of the 328 DE genes showed distinct clusters of HV and
   PI-ME/CFS samples (Fig. [295]9f, g). DE genes upregulated in the
   PI-ME/CFS females were enriched in growth hormone receptor signaling
   and ubiquitin transferase function (Fig. [296]9j). Downregulated genes
   were involved in fatty acid oxidation and mitochondrial processes
   (Fig. [297]9k). A subset of these downregulated fatty acid oxidation
   genes was highly inter-connected (Supplementary Fig. [298]S18B).
   Skeletal fatty acid oxidation is regulated during exercise as fatty
   acid uptake, is increased after moderate exercise^[299]24,[300]25, and
   is downregulated in muscle deconditioning^[301]26. These results
   suggest sex differential in the muscular bioenergetics and muscular
   deconditioning of PI-ME/CFS participants^[302]27.

Fig. 9. Male and female cohorts have distinct differential gene expression
profiles in the muscle.

   [303]Fig. 9
   [304]Open in a new tab

   a, b PCA computed from all gene expression values in samples
   highlighted from the indicated groups: HV (blue) and PI-ME/CFS (red) or
   males (turquoise) and females (orange) for the indicated PCs. c Venn
   diagram showing common DE genes identified from male and female cohorts
   (DE genes are genes with p value < 0.05 using an unadjusted two-sided
   moderated t-test). DE analysis using an unadjusted two-sided moderated
   t-test on the (d, e, h, i) male cohorts and (f, g, j, k) female
   cohorts. d, f PCA plot computed from DE genes shows robust clustering
   of samples based on the PI-ME/CFS status. e, g Volcano plots shows log
   transformed statistically significant (unadjusted p-values < 0.05 using
   an unadjusted two-sided moderated t-test) up (red) and down regulated
   (blue) genes in PI-ME/CFS male and female cohorts, respectively.
   Pathway enrichment plot of DE genes (h) upregulated and (i)
   downregulated in PI-ME/CFS male cohort. Pathway enrichment plot of DE
   genes (j) upregulated and (k) downregulated in PI-ME/CFS female
   cohorts. The top 15 pathways are labeled on the y axis and the color of
   the circle is scaled with –log-10 p-value and the size of the pathway
   circles inside the plot are proportional to the number of genes that
   overlapped with the indicated pathway. Fisher’s exact test was used in
   the ‘clusterProfiler’ R package to obtain the log-transformed p-values.
   Red color nodes: upregulated in PI-ME/CFS and blue color nodes:
   downregulated in PI-ME/CFS. DE differentially expressed, PC principal
   component. Source data are provided as a Source Data file.

Lack of differences in lipidomics between PI-ME/CFS and healthy volunteers

   Univariate analysis of the plasma lipidomic data did not identify
   statistically significant differences between HV and PI-ME/CFS groups
   (Supplementary Data S[305]20). Multivariate analysis in all
   participants as well as in male and female cohorts separately
   identified several lipids as important variables in prediction
   (Supplementary Fig. [306]S19A–C) and are consistent with a prior
   lipidomic analysis^[307]28.

Differential fecal microbiota in PI-ME/CFS by shotgun metagenomics

   Differences in the alpha diversity of stool samples (Supplementary
   Fig. [308]S20A, B, D) were noted with HVs having a greater number of
   taxa within the samples (Number of Observed Features, p = 0.002) but no
   differences in average proportional abundance (Inverse Simpson Index,
   p = 0.79). Beta diversity, as measured by Bray–Curtis dissimilarity
   (Supplementary Fig. [309]S20C), demonstrated significant differences in
   microbial community composition between the groups (PERMANOVA
   p < 0.0081).

Validation of diagnosis and course of illness on longitudinal follow-up

   Within four years of participation, four of the 17 PI-ME/CFS
   participants had a spontaneous full recovery. No other new medical
   diagnoses were reported by the PI-ME/CFS participants.

Discussion

   This study obtained a more extensive set of biological measurements in
   people with PI-ME/CFS than any previous study. Although the number of
   study subjects was small compared to the prior literature, it
   identified biological alterations and confirmed some previously
   reported biological alterations.

   All patients had documentation of good health followed by an episode of
   infection that led to ME/CFS symptoms. All PI-ME/CFS participants
   reported clinically substantial fatigue, physical symptoms, and
   decreased functional status that was 1.5 to two standard deviations
   worse than the general population. Cases were reviewed by a panel of
   adjudicators that had to unanimously agree on the case validity to be
   included in this cohort. Only 10% of those with completed reviews were
   adjudicated as PI-ME/CFS cases. Even in the cases that met study
   criteria, diagnostic misattribution was noted in 20%, with underlying
   causes becoming manifest over time. This misclassification bias has
   important ramifications on the interpretation of the existing ME/CFS
   research literature^[310]8.

   Another challenge to the characterization of ME/CFS is concerns about
   the validity of the manifestations due to depression or
   anxiety^[311]29. Our cohort underwent substantial performance validity
   testing using neuropsychological measures and demonstrated consistency
   of response, suggesting that their symptoms were reliable and a true
   representation of their disease. Even though PI-ME/CFS participants
   endorsed more depressive and anxiety symptoms than HVs, they did not
   meet psychiatric diagnostic criteria. There was also no difference in
   psychiatric history or reporting of traumatic events between the two
   groups. Thus, psychiatric disorders were not a major feature in this
   cohort and did not account for the severity of their symptoms.

   We first determined the physiological basis of fatigue in PI-ME/CFS
   participants. The notion of fatigue, as we use it, is a limit on
   ability or a diminution of ability to perform a task. Effort preference
   is how much effort a person subjectively wants to exert. It is often
   seen as a trade-off between the energy needed to do a task versus the
   reward for having tried to do it successfully. If there is developing
   fatigue, the effort will have to increase, and the effort:benefit ratio
   will increase, perhaps to the point where a person will prefer to lose
   a reward than to exert effort. Thus, as fatigue develops, failure can
   occur because of depletion of capacity or an unfavorable preference.

   There were no differences in ventilatory function, muscle oxygenation,
   mechanical efficiency, resting energy expenditure, basal mitochondrial
   function of immune cells^[312]30, muscle fiber composition, or body
   composition supporting the absence of a resting low-energy state.
   However, substantial differences were noted in PI-ME/CFS participants
   during physical tasks. Compared to HVs, PI-ME/CFS participants failed
   to maintain a moderate grip force even though there was no difference
   in maximum grip strength or arm muscle mass. This difference in
   performance correlated with decreased activity of the right
   temporal-parietal junction, a part of the brain that is focused on
   determining mismatch between willed action and resultant
   movement^[313]31. Mismatch relates to the degree of agency, i.e., the
   sense of control of the movement^[314]32. Greater activation in the HVs
   suggests that they are attending in detail to their slight failures,
   while the PI-ME/CFS participants are accomplishing what they are
   intending. This was further validated by measures of peripheral
   muscular fatigue and motor cortex fatigue^[315]20,[316]33 that
   increased only in the HVs. Thus, the fatigue of PI-ME/CFS participants
   is due to dysfunction of integrative brain regions that drive the motor
   cortex, the cause of which needs to be further explored. This is an
   observation not previously described in this population.

   Additionally, the results suggest the impact of effort preference,
   operationalized by the decision to choose a harder task when
   decision-making is unsupervised and reward values are held constant, on
   performance. 32–74% of the variance in time to grip failure for the
   PI-ME/CFS participants correlated with effort preference, which was not
   seen in HVs. This was accompanied by reduced brain activation in the
   right temporal-parietal area in PI-ME/CFS participants. Interviews with
   PI-ME/CFS participants revealed that sustained effort led to
   post-exertional malaise^[317]34. Conscious and unconscious behavioral
   alterations to pace and avoid discomfort may underlie the differential
   performance observed^[318]35.

   We measured peripheral fatigue (high:low ratio) and central fatigue
   (post exercise depression). Both types of fatigue were seen in the HVs
   but not in the PI-ME/CFS participants. Moreover, testing of effort
   preference and the participants’ own words (Supplementary Information,
   p.10) are consistent with this finding. Together these findings suggest
   that effort preference, not fatigue, is the defining motor behavior of
   this illness.

   Consistent with this observation, with strong encouragement during
   CPET, all but one of the PI-ME/CFS participants reached a respiratory
   exchange ratio of 1.1. The PI-ME/CFS group showed a clear reduction in
   cardiorespiratory fitness by reaching the AT at a lower level of work
   and ventilation^[319]36. At maximal performance, a substantial group
   difference in cardiorespiratory capacity became apparent, which was
   related to both chronotropic incompetence and physical deconditioning.
   These observations provide clarity to previous studies with
   inconsistent results^[320]37. Thus, incorporation of measures of
   autonomic tone and physical condition into studies of exercise
   performance is critical to understand the mechanisms of diminished
   cardiorespiratory function in ME/CFS^[321]38.

   A frequent complaint in our PI-ME/CFS cohort was cognitive dysfunction.
   This did not correlate with anxiety or depression measures. Standard
   clinical laboratory tests, brain imaging, measures of brain injury, and
   sleep architecture were unremarkable. Neuropsychological testing showed
   that even though the HV and PI-ME/CFS participants started with
   different levels of perceived mental and physical fatigue, there were
   no differences in cognitive performance. Further, both cognitive
   performance and perceived fatigue changed at the same rate during
   testing in both groups. This is consistent with the absence of a
   homogenous cognitive deficit in ME/CFS. Previous studies suggest a
   small, heterogenous deficit in this population^[322]39 which may not be
   evident in our study due to the small sample size. Performance validity
   testing is not routinely performed in the literature; inclusion of
   invalid performances would also bias studies toward differential
   performance.

   Taken together, this evidence suggests that physical and cognitive
   fatigue may be mechanistically different. Interestingly, PI-ME/CFS
   participants’ catechol levels in cerebrospinal fluid correlated with
   grip strength and effort preference, and several metabolites of the
   dopamine pathway correlated with several cognitive symptoms. This
   suggests that central nervous system catechol pathways are dysregulated
   in PI-ME/CFS and may play a role in effort preference and cognitive
   complaints^[323]40. The pattern suggests decreased central
   catecholamine biosynthesis in PI-ME/CFS. Similarly, decreased serum
   catechols and their metabolites have recently been reported in Long
   COVID-19;^[324]41,[325]42 testing of cerebrospinal fluid has not yet
   been reported. Autonomic testing revealed HRV measures consistent with
   an increase in sympathetic and a decrease in parasympathetic activity
   in PI-ME/CFS, and a decreased baroslope and prolonged pressure recovery
   after Valsalva consistent with previous observations of autonomic
   dysfunction in ME/CFS^[326]6,[327]43.

   We investigated several additional biological functions in PI-ME/CFS.
   Previous studies have implicated abnormalities in immunity,
   mitochondrial function, reduction-oxidation regulation, or altered
   microbiome structure in this condition^[328]1,[329]3,[330]44,[331]45.
   There were increased naïve B-cells and decreased switched memory
   B-cells in blood of PI-ME/CFS participants. However, contrary to prior
   published work^[332]46, there was no consistent pattern of autoimmunity
   across all PI-ME/CFS participants and autoantibody discovery assays did
   not reveal previously undescribed antibodies. PD-1, a marker of T-cell
   exhaustion and activation, was elevated in the cerebrospinal fluid of
   PI-ME/CFS participants. Although NK cell function was not different
   between groups in blood, they showed decreased expression of a
   cytolytic function marker in the spinal fluid. Previous studies suggest
   that NK cell function is decreased in ME/CFS^[333]47–[334]51, which may
   not be evident in our study due to the small sample size.

   Autonomic measures revealed an increase in sympathetic and a decrease
   parasympathetic activity in PI-ME/CFS that cannot be attributed to
   depression or anxiety, which is consistent with previous
   observations^[335]6,[336]43. There was also distinct gene expression,
   immune cell populations, metabolite, and protein profiles in male and
   female PI-ME/CFS participants. In the male PI-ME/CFS cohort, PBMC gene
   expression profiling showed perturbations in the T-cell activation,
   proteasome and NF-kB pathways. Analyses of serum and cerebrospinal
   fluid proteins identified several molecules related to innate immunity
   suggesting a distinct expression pattern in male cohorts. In contrast,
   in the female cohorts, gene expression profiling of PBMCs identified
   perturbations in B-cell and leukocyte proliferation processes with a
   corresponding identification of plasma lymphotoxin α1β2, which may act
   as a proliferative signal in secondary lymphoid tissues. Additionally,
   elevated levels of plasminogen in serum and eosinophil protein
   galactin-10, the C-C motif chemokine (MDC), and the IL 18 receptor
   accessory protein were present in cerebrospinal fluid of female
   PI-ME/CFS participants. Plasminogen and lymphotoxin α1β2 are known to
   activate proinflammatory states via NF-kB^[337]52,[338]53. Due to the
   small sample size of our study, we further confirmed sex differences in
   a previously published data set^[339]54. Notably, only 2% of the
   differentially expressed elements overlapped between the sex-separated
   cohort. This suggests that different immunological phenotypes may
   distinguish the PI-ME/CFS phenotype based on sex. The cause of immune
   dysregulation is not clear but may suggest the possibility of
   persistent antigenic stimulation^[340]55. Analysis of the gut
   microbiome showed differences in alpha and beta diversity consistent
   with previous studies^[341]3,[342]56. Their potential role in
   modulating the immune profile needs further exploration.

   Sex-driven group differences were also observed in gene expression
   profiles of muscle. Male PI-ME/CFS participants had upregulation of
   fatty acid beta-oxidation genes and down regulation of TRAF and
   MAP-kinase regulated genes. The female PI-ME/CFS participants had
   downregulation of fatty acid metabolism and mitochondrial processes in
   muscle. Thus, both males and females PI-ME/CFS samples had increased
   oxidative stress, but distinct pathways were perturbed in both groups.

   Metabolomics of cerebrospinal fluid identified downregulation of
   tryptophan metabolites in the PI-ME/CFS cohort, consistent with prior
   ME/CFS and Long COVID-19 studies^[343]41,[344]57–[345]59. In female
   cohorts, tryptophan, butyrate, and tricarboxylic acid related compounds
   were identified as important variables in classifying HV and PI-ME/CFS
   groups. In male cohorts, a different subset of metabolites was
   identified as important classification predictors. These findings need
   to be validated in larger PI-ME/CFS cohorts.

   This study has several limitations. This was a cross-sectional,
   exploratory case-control study. These data assess correlation, not
   causality. The sample size of the cohort was small but was very
   carefully characterized and was adequate for capturing group
   differences, noting important negative findings, and looking for
   patterns of consilience between measures. Consilience was noted among
   the multiple autonomic measures suggestive of decreased parasympathetic
   activity in PI-ME/CFS, among flow cytometry and RNA sequencing measures
   of adaptive immunity, and in the impact of sexual dimorphism across
   immunologic and metabolomic measurements. The small sample size may
   impact the precision of effect size measures, with a propensity to
   inflate estimates^[346]60. Post-hoc calculations of the effect size for
   a phenotyping sample of 21 and 17 participants to achieve a power of
   80% is 0.94, suggesting only large effects will be noted to be
   statistically significant. Overall, the groups are well-matched on
   demographic factors, but individual HV and PI-ME/CFS participants were
   not always perfectly matched. Even though a major effort was made to
   minimize misattribution, we cannot completely exclude this possibility.

   While we have focused on the positive results above, there were many
   results that were not different between HV and PI-ME/CFS participants
   (Supplementary Data S[347]22). While it is possible that small
   differences in the groups may be demonstrable in larger cohorts, these
   negative results demonstrate that these findings are not required for
   the PI-ME/CFS phenotype and are poor targets for clinical intervention.

   Considering all the data together, PI-ME/CFS appears to be a centrally
   mediated disorder. We posit this hypothetical mechanism of how an
   infection can create a cascade of physiological alterations that lead
   to the PI-ME/CFS phenotype (Fig. [348]10). Exposure to an infection
   leads to concomitant immune dysfunction and changes in microbial
   composition. Immune dysfunction may be related to both innate and
   adaptive immune responses that are sex dependent. One possibility is
   that these changes are related to antigen persistence of the infectious
   pathogen^[349]61–[350]63.

Fig. 10. Pathophysiology of PI-ME/CFS.

   [351]Fig. 10
   [352]Open in a new tab

   Diagram illustrates potential mechanisms and a cascade of events that
   lead to the development of ME/CFS after an infection. Exposure to an
   infection leads to concomitant and persistent immune dysfunction and
   changes in gut microbiome. Immune dysfunction affects both innate and
   adaptive immune systems that are sex dependent. We hypothesize that
   these changes are driven by antigen persistence of the infectious
   pathogen. These immune and microbial alterations impact the brain,
   leading to decreased concentrations of metabolites which impacts brain
   function. The catecholamine nuclei release lower levels of catechols,
   which impacts the autonomic nervous system and manifests with decreased
   heart rate variability and decreased baroreflex cardiovascular
   function, with downstream effects on cardiopulmonary capacity. Altered
   hypothalamic function leads to decreased activation of the
   temporoparietal junction during motor tasks, suggesting a failure of
   the integrative brain regions necessary to drive the motor cortex. This
   decreased brain activity is experienced as physical and psychological
   symptoms and impacts effort preferences, leading to decreased