What is still open in ME/CFS, and the experiments that would close it
We re-analysed more than 30 public ME/CFS datasets (genetics, blood chemistry, gut microbiome, immune cells, proteins, national health records) and checked every finding against a cohort it wasn't discovered in. Most popular leads failed. This page is about the ones that didn't: what is known, what is still genuinely unanswered, and the specific experiment for each.
Not medical advice and not peer reviewed. Effect sizes are group averages and diagnose no one. Our rules: people, not samples or visits, are the unit of analysis; results are corrected for multiple testing; a finding counts as replicated only if it appears in an independent cohort.
Open questions
Ordered by how much a single well-designed study could settle. For each: what the data already show, what is still unknown, and the experiment.
Is there a circulating factor that impairs healthy tissue? Unresolved
The best-powered test (Ryback 2026, 120 people, preregistered) found no effect of patient serum on healthy myoblast respiration. But it tested one readout, in undifferentiated cells, on resting blood from mostly mild and moderate patients (6 of 67 severe). Two other lines of evidence are untouched by it: patient serum weakens contracting 3D muscle (IBEC 2025, n=4), and purified patient IgG fragments endothelial mitochondria, an effect that disappears when the antibody is cleaved (Prusty lab 2026, 39 patients with MS controls).
Why is blood volume low, and is the marrow involved? Partly replicated
Red-cell volume is low in 5 isotope studies, stroke volume is low, and the heart is small. Finnish national lab records suggest a cause: people with postviral fatigue have fewer young red cells (reticulocytes 1.28% vs 1.83%) and lower soluble transferrin receptor with normal or high iron saturation, a pattern absent in 9 comparison diagnoses. That points to reduced red-cell production that isn't iron deficiency. Blood-volume hormones (renin, natriuretic peptides, EPO) look normal in proteomics.
Does low CO2 on standing drive the brain blood-flow fall? Partly replicated
Cerebral blood flow falls on tilt (26% vs 7% in controls, n=429), even without POTS. Five groups report breath CO2 falling on standing, which narrows brain vessels. In small New York Medical College studies, phenylephrine reversed the low CO2, the flow drop and the working-memory loss together. Compression halved the cardiac-output fall in a Dutch study (n=16).
Is the pituitary part of the mechanism? Partly replicated
DecodeME's risk genes are active in brain neurons and the pituitary, not immune cells, and this polygenic signal replicates in the Million Veteran Program (genetic correlation 0.73) and FinnGen. Low cortisol is reported by 19 groups vs 5 reporting high. In Finland, adrenal insufficiency and hypopituitarism are 7 to 13 times over-represented. The posterior pituitary (vasopressin, thirst, water balance) has barely been studied.
What causes the aromatic-lactate deficit? Replicated
Four related compounds (phenyllactate, 4-hydroxyphenyllactate, indolelactate, imidazole lactate) are lower in patients across 5 cohorts and 2 lab platforms (−0.53 SD, p=4×10⁻¹¹), after adjusting for drugs, diet and kidney function. The amino acids and keto acids they come from are normal. In 231 people with paired stool and blood, gut bacteria predicted them not at all. This is a host step that nobody has named as a class.
Do EPHB1 and CLEC3B cause risk, and how? Partly replicated
Of about 2,500 plasma proteins tested by Mendelian randomisation, genetically higher EPHB1 (OR 1.18) and CLEC3B/tetranectin (OR 1.21) raise risk in DecodeME and replicate in the Million Veteran Program (1.29 and 1.44). EPHB1 drives pain sensitisation through NMDA receptors, which fits the genetic overlap with chronic pain (correlation 0.45).
Is a herpesvirus still active, or did it leave an immune imprint? Partly replicated
In Finnish records, viral infections are 2 to 6 times more common before ME/CFS; bacterial ones are not. Genetically higher EBV DNA load raises risk in the Million Veteran Program (OR 1.59). Severe patients carry more EBV-matched CD8 T-cell clones (0.88% vs 0.45%, p=0.06) and fewer CMV-driven adaptive NK cells. Blood PCR is usually negative, so the question is whether reactivation happens in tissue or intermittently.
Is the nervous system stuck in an aroused state? Unreplicated
The NIH deep-phenotyping study found lower catecholamine metabolites in spinal fluid. Monocyte adrenergic-receptor expression is up in one cohort. Clinically, sleep studies in severe patients often show long sleep latency and hours awake with normal deep sleep, and crashes can follow noise, conversation or startle as well as physical effort. Nobody has measured central and peripheral noradrenaline in the same people across a crash.
Smaller open leads (5)
- Gut bloom suppressing IPA: R. gnavus, F. plautii and R. lactatiformans overgrowth explains the whole IPA gap and replicates (14/17 species). Genetics says IPA isn't causal, so this is a symptom lead. Test with competition cultures and paired stool/plasma. Guo 2023
- Collagen turnover and hypermobility: hydroxyproline and galactosyl-hydroxylysine up (+0.52 SD, 4/4 cohorts); Ehlers-Danlos and IgG subclass deficiency over-represented in Finland. Test Beighton score, IgG1–4, CTX-I and PIIINP in one cohort.
- Vessel-wall proteins lower (ITGAV, NOTCH3, TGF-β set) in 4 cohorts and long COVID. Relate to orthostatic stroke volume.
- Brain lactate raised on 7T (Oxford, 24 patients) and earlier 3T work. Resting muscle chemistry normal. Scan across a crash.
- Lymphocyte mitochondrial RNA raised in single-cell data (d up to +0.85), not replicated in bulk. Check with fresh samples.
The serum question, checked in detail
Three results seem to disagree: Fluge 2016 found patient serum raised muscle-cell respiration, Ryback 2026 found nothing in 120 people, and IBEC 2025 found serum weakened engineered muscle. We read all three in full and recomputed Ryback's result from its published per-person data.
One specific result
Resting serum from mostly mild-to-moderate female patients does not change maximal respiration in healthy myoblasts. Effect +2.5 pmol/min (95% CI −1.6 to 6.6). From their per-person data we get d=0.14 (−0.22 to 0.50), which excludes Fluge's d=1.32. The study was preregistered, blinded and randomised, and it is the most rigorous in the field.
Fluge's original arm was weaker than it looked: 12 vs 12, no cell-count correction, and replicate wells probably treated as independent samples.
Most of the serum hypothesis
- Severe disease. Only 6 of 67 were severe (Fluge: 9 of 12). In those 6, d=0.00 with a confidence interval of −0.84 to +0.85: uninformative.
- Post-exertional state. All samples were taken at rest by design.
- Other readouts. No force, lactate, PDK, morphology or gene expression.
- Detection floor. No spike-in positive control, and plates explained 42% of variance.
Their abstract says "strong evidence against"; their discussion says the results "do not rule out the possibility" of serum factors.
Why Ryback and IBEC can both be right
| Ryback 2026 | IBEC (Mughal) 2025 | |
|---|---|---|
| Cells | Undifferentiated primary myoblasts, flat dish | Differentiated myotubes in 3D, anchored to pillars |
| Readout | Oxygen consumption only | Contractile force; RNA-seq; mitochondrial shape |
| Serum dose and time | 20% for 6 days, one time point | 5% at 48, 96 and 144 h |
| Timing effect | Measured at 144 h | Respiration up at 48 h, then atrophy and fragmented mitochondria by 96–144 h |
| Patients | 67 community, mostly mild–moderate | 4 from a tertiary clinic, fasting morning draw |
| Rigour | Preregistered, blinded, 1,926 wells | Not preregistered; n=4, tissues as replicates |
The early respiration rise IBEC saw at 48 h is Fluge's direction, but it rests on 3 sera per group, too few to weigh against Ryback. The force loss is the independent result, and it needs replicating at scale.
Other serum and plasma effects
- Prusty lab 2026: purified patient IgG fragments endothelial mitochondria; lost on Fab/Fc cleavage. 39 ME/CFS, 20 MS controls. The strongest current candidate.
- Bertinat 2022: patient plasma lowers nitric oxide in vessel cells (11 vs 12).
- Esfandyarpour 2019: nanoneedle impedance; plasma-swap never independently replicated.
- Cervia-Hasler 2024: complement dysregulation in long COVID. Neither Fluge nor Ryback heat-inactivated serum, so complement was present in both.
- Appelman 2024: long-COVID muscle damage worsens after induced exertion, which is why post-exertion sampling matters.
What would settle it
- At least 30 severe patients with home blood draws, at Ryback's level of rigour.
- Paired samples from the same person at rest and 24 h after exertion.
- A spike-in positive control so every assay states the smallest effect it could detect.
- Differentiated myotubes read at 24, 48, 96 and 144 h with a force readout.
- Fractions (IgG, heat-labile complement, vesicles) rather than 20% whole serum.
What severe, recent-onset cases add
Most ME/CFS data come from mild-to-moderate patients years into illness who can travel to a clinic. Severe and bed-bound patients rarely appear: 6 of 120 in the key serum study, and none in most treatment trials. The first years after a viral trigger are also rare in datasets, though that is when mechanisms are most likely to be active. Things that show up in severe, early cases and deserve study:
Crashes from non-physical load
Noise, conversation, startle or a hair wash can trigger post-exertional malaise. Exercise tests are unsafe for these patients and miss this, so PEM models need other provocations.
A falling threshold
Each crash can lower the threshold for the next. Nobody has tracked this with objective data, though consumer wearables already record resting heart rate and HRV continuously.
Sleep that is hard to start, not shallow
Long sleep latency and hours awake at night, with normal or high deep sleep. That fits hyperarousal more than a sleep-architecture defect.
Water and salt handling
Heavy thirst, dilute urine and a strong response to IV saline raise questions about vasopressin and blood volume that routine tests don't answer. Copeptin is rarely measured.
Many changes at once
Sick patients often start several drugs within days. That makes it impossible to learn what helped. One change at a time, with a wearable-based endpoint, turns care into usable n-of-1 data.
Samples that are never taken
Blood from a crash day, from a bed-bound patient, or before a steroid or hormone is started is almost never banked. It is exactly what the open questions above need.
A plan for closing the open questions
The bottleneck is not ideas. It is assays with a known detection floor, blood drawn at the right moment, severe patients reached at home, and designs where each person is their own control.
Qualify the assays before using patient samples
Run healthy sera across plates, cell lots, operators and freeze-thaw cycles for each readout: 3D muscle force, respiration, blinded mitochondrial morphology, endothelial IgG assay. Add a spike-in positive control. Report the smallest effect each assay can detect, and preregister.
One cohort, sampled at the right moments
- Blood at a stable baseline and during a crash (or 24 h after a standard provocation for those who can tolerate it), processed identically and banked in aliquots.
- Reticulocytes, sTfR, EPO, hepcidin, copeptin, cortisol/ACTH, IgG subclasses, noradrenaline, and blood volume where feasible.
- Graded head-up tilt with capnography and, where possible, transcranial Doppler.
- Chiral aromatic-lactate panel, paired stool, genotype for DecodeME and pQTL variants, EBV-lytic T-cell responses.
- Continuous wearable heart rate and HRV for the whole period.
Blinded two-lab serum transfer
The same coded sera go to a 3D-muscle lab and an endothelial lab, each with one preregistered endpoint. Compare crash vs baseline within person and severe vs mild. If a signal clears the detection floor, fractionate (IgG, complement, vesicles) to find it.
Mechanism follow-ups
- Low red-cell production: erythroid progenitor counts and EPO response in vitro.
- Pituitary: dynamic testing in those with borderline results.
- Aromatic lactates: isotope tracing and D/L chirality.
- EPHB1: colocalisation, CSF levels, sensory testing by genotype.
- Low CO2: crossover of compression, midodrine and CO2 clamping during tilt.
Small, well-measured treatment trials
Randomised crossovers or n-of-1 series with objective endpoints: wearable resting heart rate, upright CO2, step counts, a cognitive task. Candidates if the physiology holds: volume expansion, compression, midodrine, pyridostigmine, and targeted hormone replacement for proven deficiency. Follow the daratumumab RCT.
Open data from day one
Per-person data with IDs that link across modalities, deposited on publication. Ryback's per-person file is why we could check the severe subgroup here. Several other leads are stuck because data sit behind requests.
What we tested and consider settled
These were tested with enough data to matter and did not hold up. They are listed briefly so effort goes elsewhere.
Serum raising myoblast respiration at rest
Refuted in mild–moderate patients (Ryback 2026). Severe and post-exertion samples remain open.
Metabolites as causes
Mendelian randomisation is null for IPA, indolelactate, kynurenine, ketones, carnitine and bile acids.
Kynurenine up
It is lower in patients across 7 blood cohorts and in spinal fluid.
Immune-cell signatures
Single-cell (503,739 cells), methylation, epigenetic clocks, B- and T-cell repertoires, interferon, Th17: nothing replicates.
Two published claims
De Vega 2017 methylation is granulocyte contamination; Sato 2021 IGHV3-30/3-33 did not replicate.
GPCR autoantibodies
Not raised in 164-person profiling; BC007 and immunoadsorption vs sham failed.
Blood gene expression
Five cohorts, 219 cases: no gene set passes correction.
Single-cohort metabolites
Fatty acids, bile acids, VMA and sphingolipids each failed across other cohorts.
Null trials
Rituximab (n=151), Paxlovid (3 trials), IVIG in POTS, low-dose naltrexone in post-COVID ME/CFS, CoQ10 alone.
Data we used
All public. Per-person data from severe or recent-onset patients would make the most difference.
| Source | What | Size |
|---|---|---|
| DecodeME | GWAS summary statistics | 15,579 cases |
| FinnGen R12 / Risteys | Postviral fatigue GWAS, comorbidities, lab values | ~1,600 cases |
| BioMapAI (Xiong 2025) | Gut metagenome, plasma metabolome, immune, clinical | 153 patients, 96 controls |
| NIH (Walitt 2024) | Tilt, CSF, flow, proteomics | 17 patients, 21 controls |
| Metabolomics Workbench | Naviaux, Nagy-Szakal, Che, NIH CSF, Japanese cohorts | 9 studies |
| Ryback 2026 | Per-person myoblast respiration | 67 patients, 53 controls |
| GEO | Single-cell, bulk RNA, methylation | 12 datasets |
| PRIDE and supplements | SomaScan and mass-spec proteomics | 7 datasets |
| ClinicalTrials.gov | Trials with posted results | 165 trials |
Out of reach so far: participant-linked NIH tables, Hoel 2021 per-person metabolomics, full plasma pQTL statistics, individual Finnish registry data.