This is the first description of the effects of exercise on cerebrospinal fluid miRNA expression in healthy subjects. Exercise diminished miR-328 and miR-608 in all subjects suggesting a general effect on the brain (Fig. 8, Supplementary Table S5). Exercise caused distinct patterns of miRNA changes in CFS and the START and STOPP phenotypes of GWI indicating significant pathophysiological differences between conditions.

Unlike our starting hypothesis, there were no differences in miRNA levels between the nonexercise groups of control, CFS and GWI subjects. Therefore, baseline levels of cerebrospinal fluid miRNAs may not be useful for diagnosis of CFS or GWI.

The only significant differences between groups after exercise were diminished miR-22-3p in START compared to SC and STOPP, and elevated miR-9-3p in START compared to STOPP (Fig. 6). These differences between START and STOPP support our 2 phenotypes of GWI25.

The most striking findings were the changes between post-exercise groups and their appropriate nonexercise comparison groups. SC had 5 elevated miRNAs after exercise, compared to 3 for START, 1 for STOPP, and none in CFS (Fig. 7, Supplementary Table S4).

The reduction of miR-608 after exercise has implications for the cholinergic hypothesis of GWI pathophysiology because it targets acetylcholinesterase and interleukin-6 (IL6) mRNAs. miR-608 binds weakly to the single-nucleotide polymorphism rs17228616 allele in the 3′-untranslated region of acetylcholinesterase mRNA44. Homozygotes for rs17228616 have reduced affinity for miR-608. This promotes mRNA stability and increases acetylcholinesterase protein translation. As a consequence, more miR-608 is available to bind to IL6 mRNA and reduces its translation. This allele also contributes to reduced cortisol and elevated blood pressure. Because rs17228616 promotes higher acetylcholinestase activity, it may be relatively protective against nerve agent and pyridostimine bromide exposure.

miR-let-7i-5p, miR-93-3p and miR-200a-5p were significantly diminished after exercise in START, STOPP and CFS, but not SC (Fig. 9). This was consistent with a cardinal finding in CFS and GWI: function may appear normal when rested, but will deteriorate after a physiological stressor1,2,3,4,7,25,45,46. miR-let-7i was reduced in plasma after exercise in athletes47. miR-let-7i has decreased expression in the prefrontal cortex of FSL rats in a model of depression48. IL6 is a target of miR-let-7i, and, as predicted, this cytokine was significantly elevated in the brains of these rats. When FSL rats were given access to running wheels, their miR-let-7i expression was increased and IL6 reduced. Modulation of miR-let-7i and IL-6 may contribute to exercise-induced benefits in “inflammatory” depression48. miR-let-7i also contributes to the regulation of acetylcholine’s muscarinic and α4β2 nicotinic receptors and epigenetic regulation of acetylcholinesterase. These animal models may not be appropriate for CFS or GWI because human subjects develop exertional exhaustion after exercise, and are unlikely to significantly increase spontaneous exercise levels when provided with a treadmill45.

The CFS group had 12 miRNAs reduced after exercise. miR-186-3p was decreased in aging mice49 where it targets β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA. Diminished miR-186-3p allows increased BACE1 mRNA translation and cleavage of amyloid peptides that increase the risk for brain disease. miR-19b-3p was reduced in serum from Alzheimer’s patients, and targeted signal transduction and activator of transcription 3 (STAT3) mRNA in a murine model50. miR-92a-3p was increased in glioblastoma and targeted BCL2L11 to reduce tumor apoptosis51. Its reduction after exercise may promote apoptosis in CFS. miR-126-5p was highly expressed in endothelial cells where it targets vascular (VCAM), intercellular (ICAM) and activated leukocyte (ALCAM) cell adhesion molecule mRNAs and so reduces transendothelial migration52,53. This is relevant for immune cell influx into the brain, and hypotheses of neuroinflammation in CFS pathogenesis.

Neurons may be the sources of miR-124-3p, miR-127-3p, miR-433, and miR-323b-5p (Figs 4 and 10)54. There was little overlap with the miRNAs synthesized in astrocytes, oligodendrocytes and microglia.

The choroid plexus epithelium may be a primary source of miRNAs in cerebrospinal fluid55,56,57. Epithelial cells form a monolayer linked by tight junctions that creates the “shrink wrapped” cellular barrier around fenestrated capillaries58. Interferon-gamma and other mediators generated by exercise, inflammation, and other stressors act directly on choroid plexus to modulate barrier permeability, plasma protein transport, protein synthesis and secretion of nutrients into cerebrospinal fluid59,60,61. miR-328, which was present in all subjects and reduced after exercise (Fig. 8), binds to the 3′-untranslated regions of CD44 and collagen type 1α1 mRNAs to modulate extracellular barrier functions62. Choroid plexus miRNAs55 are packaged into extracellular vesicles and released into cerebrospinal fluid56,57,63,64. Downstream targets include subventricular neural stem cells, mature neurons, astrocytes, oligodendrocytes, microglia, meningeal and central immune cells56,57,63,65,66,67. Blockade of extracellular vesicle secretion from choroid plexus cells decreased brain inflammation in a mouse model of lipopolysaccharide-induced inflammation56. Choroid plexus miRNAs may be novel drug targets to modulate acute illness behaviours, fever, and chronic pain in systemic illnesses.

Choroid plexus is dysfunctional in Alzheimer’s disease68. This provides the rationale to consider the role of the blood – cerebrospinal fluid barrier in the cognitive dysfunction of CFS and GWI. There are numerous reports of elevated and diminished miRNAs in cerebrospinal fluid in Alzheimer’s disease36,69,70,71,72,73,74, but none matched the patterns of our groups. miR-let-7i-5p36 was elevated in Alzheimer’s, but levels were equivalent in nonexercise groups (Fig. 8).

Depression is in the differential diagnosis because of the shared ancillary diagnostic criteria (Fig. 1)75,76,77,78. Major depressive disorder is defined by affective dysfunction with sadness, flat affect and anhedonia as essential features, followed by secondary criteria including fatigue, cognitive, sleep, and somatic dysfunction78. However, screening questionnaires for depression emphasize somatic symptoms79,80. Complaints of fatigue, sleep, and cognitive dysfunction will inflate total questionnaire scores, and may lead to false positive inference of major depressive disorder even if anhedonia or affective complaints are absent81,82,83. This is particularly problematic in CFS and GWI where these features are diagnostic criteria (Fig. 1). As a result, Center for Epidemiology – Depression (CESD)33 scores were significantly elevated in GWI (78.3%), CFS (64.0%) and control (25.0%) groups (Table 1).

Quantitative PCR of miRNAs offers a more objective solution84. miR-16 in cerebrospinal fluid was significantly lower in major depressive disorder patients than control subjects85. However, this was not confirmed in an independent group who had a different pattern of 11 significantly elevated and 5 reduced miRNAs86. Our data did not confirm either of these findings because only 3 of the miRNAs were detected with Ct ≤ 35 in more than two thirds of our nonexercise group. miR-425-3p was significantly reduced in depression patients84,85,86, and was detected in about half of all nonexercise subjects. It was increased after exercise in SC, START and STOPP but not CFS (Fig. 7). This lack of reproducibility highlights the need to independently verify miRNA findings, and supports our rationale for strict statistical criteria to define potential miRNA biomarkers.

The pain and tenderness of GWI subjects (Table 1) indicated systemic hyperalgesia and suggested parallels with fibromyalgia34. Nine miRNAs were virtually undetectable in 10 fibromyalgia women compared to 8 healthy control women87. miR-99b-5p and miR-29a-3p were absent in fibromyalgia, but were detected in more than two thirds of our participants. miR-99b-5p was significantly increased after exercise in SC, START and STOPP (Fig. 7). The other 7 miRNAs were detected in less than half of our specimens. This suggested that GWI and CFS were distinct from fibromyalgia.

Limitations to the diagnostic use of quantitative miRNA analysis in cerebrospinal fluid include the remarkable lack of consensus about miRNA levels in control subjects. This can be remedied by standardization of reagents and protocols72, open source sharing of study outcomes, and meta-analysis of the raw data. The yield of extracted miRNA88 and detectability were improved with 0.5 ml instead of 0.2 ml of cerebrospinal fluid36,87. QPCR with Ct cut-offs ≤ 35 cycles reduced amplification artifacts73,87. The wide range of miR-22-3p Ct values (Fig. 6) may be due to commercial changes in reagents designed to improve miRNA detection. Highly abundant miRNAs that were detected with Ct < 35 in all subjects were used as normalizers (Fig. 4) rather than the global average level, miR-423-5p, miR-124-3p or U636,71,73,86,89. Constraints included (i) significant ANOVA and Tukey HSD between groups, (ii) significant FDR to correct for multiple comparisons, and (iii) focusing on miRNAs that were detected in more than two thirds of subjects per group that may be viable biomarker candidates for use in the general population36,71,86. Ages were comparable between groups74 (Table 1) and there were no differences in expression between males and females43,87. Next generation sequencing is an excellent discovery tool but needs careful internal standardization to be as sensitive as QPCR for quantification90,91,92. Adequate sample sizes were essential because our initial findings with about a dozen subjects per group showed differences between START and STOPP after exercise93, but these differences eventually regressed to the mean as more subjects were analyzed. This is especially pertinent to smaller studies examining the differential diagnosis of CFS and GWI85,86,87,94,95.

Limitations of the testing paradigm include the intensive nature of the exercise and magnetic resonance imaging characterization of GWI subjects to determine their phenotypes. Lumbar puncture was required to obtain the cerebrospinal fluid miRNA biomarkers, but this procedure is not a contraindication to making an objective diagnosis of GWI. On the contrary, magnetic resonance imaging with cerebrospinal fluid QPCR miRNA profiling may be complementary tools for diagnosis of CFS, GWI and their subtypes.