Pupillometry as Autonomic Biomarker [pupillometry-autonomic-biomarker-mecfs]
1 Sander et al. 2025 — Validation of Monocular Pupillometry in Autonomic Failure
Full Citation:: Sander L, Oommen G, Brophy C, Bohus-Roper S, Chiaro G, Bremner F, Iodice V. Validation of monocular pupillometry in healthy controls and patients with autonomic dysfunction: pupillary biomarkers for autonomic failure. European Journal of Neurology. 2025;32(8):e70320. (Sander et al. 2025) DOI:: 10.1111/ene.70320 PMID:: 40831073 Study Design:: Prospective validation study Sample Size:: Patients with autonomic dysfunction + healthy controls Key Findings::
- Handheld monocular pupillometer validated for autonomic screening
- PLR parameters (constriction amplitude, constriction latency, dilation velocity) distinguish autonomic failure patients from controls
- Normative values established for clinical interpretation
- Noninvasive, portable assessment tool suitable for broad clinical applicationConclusion:: Pupillometry provides validated, noninvasive ocular autonomic evaluation and is suitable as an autonomic screening tool in autonomic disorders. Limitations:: Moderate sample size; single study without independent replication; no ME/CFS-specific data. ME/CFS Relevance:: Provides validated methodology directly applicable to autonomic assessment in ME/CFS. Establishes normative PLR parameters for comparison. Certainty Assessment::
- *Quality:* High (prospective validation, top neurology journal, rigorous methodology)
- *Sample:* Medium
- *Replication:* Single study
- *Score:* 0.702 Koay et al. 2025 — Multimodal Autonomic Biomarkers Predict Phenoconversion in PAF
Full Citation:: Koay S, Vichayanrat E, Bremner F, Valerio F, Mackenzie R, Chiaro G, Ingle G, McNamara P, Watson L, Panicker JN, Lunn MP, Mathias C, Iodice V. Multimodal autonomic biomarkers predict phenoconversion in pure autonomic failure. Annals of Clinical and Translational Neurology. 2025;12(11):2170–2180. (Koay et al. 2025) DOI:: 10.1002/acn3.70140 PMID:: 40696849 Study Design:: Longitudinal multimodal biomarker study Key Findings::
- Pupillometry component of multimodal autonomic panel predicting phenoconversion from PAF to MSA/LBD
- Supports role of pupillary assessment in longitudinal autonomic monitoring
- Multimodal approach outperforms single-modality assessmentConclusion:: Pupillometry contributes to multimodal autonomic biomarker panels capable of predicting disease trajectory. Limitations:: PAF-specific population; moderate sample; pupillometry is one component of multimodal panel, not standalone. ME/CFS Relevance:: Demonstrates value of pupillometry in longitudinal autonomic assessment and phenoconversion prediction — directly applicable to ME/CFS subgroup stratification. Certainty Assessment::
- *Quality:* Medium-High (longitudinal design, multimodal approach)
- *Sample:* Medium
- *Replication:* Single study
- *Score:* 0.653 Parmak Yener et al. 2026 — ML-Based Pupillography for Autonomic Dysfunction in MS
Full Citation:: Parmak Yener N, Fırat Y, Seferoğlu M, Kargın AM, Kılıçaslan Y. Exploratory analysis of pupillography-based machine learning for assessing autonomic dysfunction in multiple sclerosis. Multiple Sclerosis and Related Disorders. 2026;112:107326. (Parmak Yener et al. 2026) DOI:: 10.1016/j.msard.2026.107326 PMID:: 42322958 Study Design:: Exploratory ML analysis of pupillographic features Key Findings::
- First ML-based analytical framework applied to pupillographic features for autonomic assessment in MS
- PLR dynamics quantified via pupillography provide informative features for autonomic dysfunction classification
- Demonstrates feasibility of automated PLR-based autonomic screeningConclusion:: Pupillography-based ML analysis is feasible for assessing autonomic dysfunction in MS and may generalize to other conditions. Limitations:: Exploratory design; moderate sample; novel methodology not yet validated; MS-specific. ME/CFS Relevance:: Establishes precedent for automated PLR-based autonomic screening applicable to ME/CFS. ML approach could enable scalable, objective autonomic assessment. Certainty Assessment::
- *Quality:* Medium (exploratory, peer-reviewed)
- *Sample:* Medium
- *Replication:* Single study with novel methodology
- *Score:* 0.504 Master et al. 2020 — PLR Metrics as Biomarker for Adolescent Concussion
Full Citation:: Master CL, Podolak OE, Ciuffreda KJ, Metzger KB, Joshi NR, McDonald CC, Margulies SS, Grady MF, Arbogast KB. Utility of pupillary light reflex metrics as a physiologic biomarker for adolescent sport-related concussion. JAMA Ophthalmology. 2020;138(11):1135–1141. (Master et al. 2020) DOI:: 10.1001/jamaophthalmol.2020.3466 PMID:: 32970102 Study Design:: Cross-sectional diagnostic biomarker study Sample Size:: n=352 adolescents (concussed and controls) Key Findings::
- PLR metrics validated as objective physiologic biomarker for concussion
- PLR distinguishes concussed adolescents from healthy controls with good diagnostic performance
- Deficits in visual accommodation and autonomic function after concussion make PLR a promising target biomarker
- Provides strong precedent for PLR as neurologic/autonomic biomarker in clinical populationsConclusion:: PLR metrics serve as an objective physiologic biomarker for adolescent concussion, supporting broader application of pupillometry in neurologic assessment. Limitations:: Concussion-specific; adolescent population; single center. ME/CFS Relevance:: Large-scale validation of PLR as objective biomarker establishes precedent and methodology transferable to ME/CFS. Demonstrates that PLR captures central autonomic dysfunction in a clinical population. Certainty Assessment::
- *Quality:* High (large sample, high-impact journal, rigorous design)
- *Sample:* Large (n=352)
- *Replication:* Single study but validates PLR as biomarker paradigm
- *Score:* 0.755 Egg et al. 2002 — Null Finding: Pupillary Unrest Not Associated with MS Fatigue
Full Citation:: Egg R, Högl B, Glatzl S, Beer R, Berger T. Autonomic instability, as measured by pupillary unrest, is not associated with multiple sclerosis fatigue severity. Multiple Sclerosis. 2002;8(3):256–260. (Egg et al. 2002) DOI:: 10.1191/1352458502ms793oa PMID:: 12120699 Study Design:: Cross-sectional pupillography study Sample Size:: n=51 MS patients, n=22 healthy controls Key Findings::
- *Null result:* Pupillary unrest index (PUI) inversely correlated with fatigue severity in MS (r = -0.521, p = 0.001) — opposite of the autonomic instability hypothesis
- Central autonomic instability as measured by pupillography does NOT explain MS fatigue
- Challenges the generic "autonomic dysfunction causes fatigue" narrative
- MS patients had significantly higher fatigue scores than controls, yet pupillary unrest was inversely relatedConclusion:: Autonomic instability, as measured by pupillary unrest, is not associated with MS fatigue severity. The relationship between autonomic function and fatigue may be more complex than simple dysautonomia. Limitations:: Older study (2002); small sample; MS-specific (may not generalize); PUI measures spontaneous pupillary oscillations, not stimulated PLR parameters. ME/CFS Relevance:: Critical negative control: the autonomic instability → fatigue hypothesis may not hold universally. If pupillary autonomic markers are pursued in ME/CFS, this finding underscores the need for disease-specific validation rather than assuming cross-disease generalizability. Certainty Assessment::
- *Quality:* Low-Medium (small sample, older, single study)
- *Sample:* Small (n=51 MS + 22 controls)
- *Replication:* Single study, not replicated
- *Score:* 0.406 Drummond and Finch 2022 — Pupillary Reflexes in CRPS: Locus Coeruleus Deficit
Full Citation:: Drummond PD, Finch PM. Pupillary reflexes in complex regional pain syndrome: asymmetry to arousal stimuli suggests an ipsilateral locus coeruleus deficit. Journal of Pain. 2022;23(1):131–140. (Drummond and Finch 2022) DOI:: 10.1016/j.jpain.2021.07.003 PMID:: 34375745 Study Design:: Cross-sectional pupillometry study with arousal-evoked and light-reflex paradigms Sample Size:: n=33 CRPS patients Key Findings::
- Pupil on CRPS-affected side was smaller than contralateral pupil at baseline and after arousal stimuli
- Pupillary light reflex (constriction to bright light, dilation in dim light) was symmetrical and intact
- Asymmetry specific to arousal-evoked dilation, implicating ipsilateral locus coeruleus (LC) deficit
- LC deficit may contribute to autonomic disturbances, auditory discomfort, and pain in CRPS
- Acoustic startle stimuli failed to inhibit shock-induced pain on affected sideConclusion:: Neural activity in LC-linked pathways is compromised ipsilaterally in CRPS, contributing to autonomic disturbances and pain amplification. Limitations:: CRPS-specific; moderate sample (n=33); single study; arousal-evoked pupillometry methodology not yet standardized. ME/CFS Relevance:: Establishes pupillometry as noninvasive probe of LC function — directly relevant to central noradrenergic deficiency hypothesis in ME/CFS (Aregawi 2026, Walitt 2024). Supports use of task-evoked pupillary dynamics to assess LC integrity in ME/CFS. Certainty Assessment::
- *Quality:* Medium (innovative methodology, good journal)
- *Sample:* Small-Medium (n=33)
- *Replication:* Single study
- *Score:* 0.557 Rizzuto et al. 2025 — Pupillary Hippus as Autonomic Biomarker
Full Citation:: Rizzuto V, Laurino M, Montanari R, Gemignani A, Figus M, Covello G, Candelise N, Borroni D, Laganovska G, Mesin L. Pupillary hippus as a biomarker: spectral signatures and complexity approaches in autonomic and clinical contexts. Bioengineering. 2025;12(12):1376. (Rizzuto et al. 2025) DOI:: 10.3390/bioengineering12121376 PMID:: 41463672 Study Design:: Narrative review with synthesis Key Findings::
- Synthesizes spectral signatures of pupillary hippus (spontaneous bilateral pupil oscillations under constant illumination)
- Low-frequency hippus components covary with respiratory/vasomotor autonomic rhythms
- Higher-frequency fluctuations sensitive to cognitive load, visual fatigue, and pathological states
- Proposes unified taxonomy of hippus frequency bands
- Identifies methodological heterogeneity as key translational gapConclusion:: Pupillary hippus provides a noninvasive window into autonomic and central nervous system dynamics, but validation and standardization are needed before clinical adoption. Limitations:: Review article (no original data); methodological heterogeneity across studies limits synthesis; hippus-based biomarkers not yet validated in clinical populations. ME/CFS Relevance:: Provides theoretical and methodological framework for using spontaneous pupillary oscillations as autonomic biomarker. Low-frequency hippus tracking autonomic rhythms could be applied to assess autonomic dysfunction in ME/CFS without requiring light stimulation. Certainty Assessment::
- *Quality:* Medium (comprehensive review, no original data)
- *Sample:* N/A (review)
- *Replication:* N/A (synthesis)
- *Score:* 0.608 Lisowski et al. 2025 — Diagnostic Significance of Pupillary Reflex Pathways
Full Citation:: Lisowski Ł, Lisowska J, Charytonowicz A, Mariak Z, Obuchowska I, Konopińska J. The diagnostic significance of pupillary reflex pathways: insights from classical examination and advanced pupillometry. Frontiers in Neuroscience. 2025;19:1677431. (Lisowski et al. 2025) DOI:: 10.3389/fnins.2025.1677431 PMID:: 41169745 Study Design:: Comprehensive interdisciplinary review Key Findings::
- Reviews anatomical pathways: afferent (retina → pretectal olivary nucleus → Edinger-Westphal nucleus) and efferent (EW → ciliary ganglion → iris sphincter M3 muscarinic; sympathetics → iris dilator alpha-1 adrenergic)
- Links pupillary reflex abnormalities to specific neurological lesion locations
- Classical examination plus automated pupillometry provides complementary diagnostic information
- Identifies pupillometry as noninvasive window into autonomic pathway integrityConclusion:: Understanding pupillary reflex pathways enables targeted neurological and autonomic diagnosis through pupillometry. Limitations:: Review article (no original data); primarily classical neurological examination focus; limited coverage of quantitative pupillometry methodology. ME/CFS Relevance:: Provides essential anatomical and physiological framework for understanding how pupillometry measures autonomic function. The M3 muscarinic and alpha-1 adrenergic pathways are the same receptors targeted by GPCR autoantibodies found in ME/CFS (Szklarski 2021, Azcue 2026). Certainty Assessment::
- *Quality:* Medium-High (comprehensive, peer-reviewed review)
- *Sample:* N/A (review)
- *Replication:* N/A (established neuroanatomy)
- *Score:* 0.659 Szklarski et al. 2021 — CD26 and GPCR Autoantibodies in ME/CFS
Full Citation:: Szklarski M, Freitag H, Lorenz S, Becker SC, Sotzny F, Bauer S, Hartwig J, Heidecke H, Wittke K, Kedor C, Hanitsch LG, Grabowski P, Sepúlveda N, Scheibenbogen C. Delineating the association between soluble CD26 and autoantibodies against G-protein coupled receptors, immunological and cardiovascular parameters identifies distinct patterns in post-infectious vs. non-infection-triggered myalgic encephalomyelitis/chronic fatigue syndrome. Frontiers in Immunology. 2021;12:644548. (Szklarski et al. 2021) DOI:: 10.3389/fimmu.2021.644548 PMID:: 33889154 Study Design:: Cross-sectional autoantibody profiling study Sample Size:: ME/CFS cohort (post-infectious and non-infection-triggered) Key Findings::
- Infection-triggered ME/CFS patients show elevated autoantibodies against alpha-1 adrenergic receptors (pupillary dilator) and M3 muscarinic acetylcholine receptors (pupillary sphincter)
- sCD26 concentrations inversely correlate with these autoantibodies, distinct patterns by onset type
- Establishes biochemical bridge between pupil autonomic control receptors and ME/CFS autoimmunity
- Non-infection-triggered ME/CFS shows different pattern linked to orthostatic regulationConclusion:: Autoantibodies against the exact receptors controlling pupillary function are elevated in infection-triggered ME/CFS, suggesting autoimmune-mediated autonomic dysfunction. Limitations:: Cross-sectional; no pupillometry measurements; single-center; does not directly assess pupil function. ME/CFS Relevance:: Critical indirect link: the receptors targeted by autoantibodies in ME/CFS (alpha-1 AR, M3 mAChR) are the same receptors that control pupillary dilation and constriction. This provides a biochemical mechanism by which pupillometry could detect autoimmune autonomic dysfunction in ME/CFS. Certainty Assessment::
- *Quality:* Medium-High (well-characterized cohort, rigorous serology)
- *Sample:* Medium
- *Replication:* Partially replicated (consistent with broader GPCR autoantibody literature)
- *Score:* 0.6510 Oesch-Régeni et al. 2025 — Plasmapheresis/IVIG in ME/CFS with M3 Antibodies
Full Citation:: Oesch-Régeni B, Germann N, Hafer G, Schmid D, Arn N. The effect on quality of life of therapeutic plasmapheresis and intravenous immunoglobulins on a population of myalgic encephalomyelitis/chronic fatigue syndrome patients with elevated β-adrenergic and M3-muscarinic receptor antibodies—a pilot study. Journal of Clinical Medicine. 2025;14(11):3802. (Oesch-Régeni et al. 2025) DOI:: 10.3390/jcm14113802 PMID:: 40507564 Study Design:: Pilot interventional study (plasmapheresis/IVIG) Sample Size:: Small pilot (ME/CFS patients with elevated M3 and beta-adrenergic autoantibodies) Key Findings::
- M3-muscarinic receptor autoantibodies are clinically actionable in ME/CFS
- Patients treated with plasmapheresis/IVIG showed quality of life improvements
- Demonstrates clinical relevance of M3 pathway in ME/CFS pathophysiologyConclusion:: M3-muscarinic receptor autoantibodies represent a treatable target in a subset of ME/CFS patients. Limitations:: Pilot study without control group; very small sample; no pupillometry measurements; uncontrolled design limits causal inference. ME/CFS Relevance:: Establishes that M3 receptor autoantibodies (which would affect pupillary sphincter function) are not only present in ME/CFS but clinically actionable. Supports rationale for pupillometry as functional readout of M3-mediated autonomic function. Certainty Assessment::
- *Quality:* Low (pilot, no control group)
- *Sample:* Small
- *Replication:* Single pilot, not replicated
- *Score:* 0.3511 Azcue et al. 2026 — GPCR Autoantibodies in Post-COVID and CFS
Full Citation:: Azcue N, Prada A, Del Pino R, Acera M, Fernández-Valle T, Ayo-Mentxakatorre N, Pérez-Concha T, Murueta-Goyena A, Lafuente JV, López de Munain A, Ruiz Irastorza G, Ribacoba L, Gabilondo I, Tijero-Merino B, Gómez-Esteban JC. Involvement of autoantibodies against G protein-coupled receptors in post-COVID condition and Chronic Fatigue Syndrome. Scientific Reports. 2026;16(1):11837. (Azcue et al. 2026) DOI:: 10.1038/s41598-026-49131-9 PMID:: 42082542 Study Design:: Large cross-sectional autoantibody profiling Sample Size:: Large cohort (post-COVID and CFS patients) Key Findings::
- GPCR autoantibodies including anti-alpha-1 adrenergic detected in both post-COVID and CFS
- Autoantibodies target receptors mediating autonomic control, including pupillary function
- Provides large-cohort evidence for autoimmune autonomic dysfunction in post-infectious syndromes
- Replicates and extends prior GPCR autoantibody findings (Szklarski 2021, Freitag 2021)Conclusion:: GPCR autoantibodies are involved in both post-COVID condition and CFS, supporting autoimmune mechanisms in post-infectious autonomic dysfunction. Limitations:: Cross-sectional; no functional pupillometry measurements; does not directly link autoantibody levels to pupillary function. ME/CFS Relevance:: Large-cohort replication of alpha-1 adrenergic receptor autoantibodies in ME/CFS and related conditions. Directly relevant to pupillometry as functional readout: if autoantibodies block or stimulate these receptors, pupillary dynamics should detect the functional consequence. Certainty Assessment::
- *Quality:* Medium-High (large cohort, peer-reviewed)
- *Sample:* Large
- *Replication:* Replicates prior GPCR autoantibody findings
- *Score:* 0.7012 Li et al. 2026 — Scotopic-Photopic Pupil Difference as Glycemic Indicator
Full Citation:: Li Y, Pan Z, Ren Q, Shi F, Wan P. The difference in scotopic and photopic pupil responses: a potential indicator for long-term glycemic management. Frontiers in Neurology. 2026;17:1785905. (Li et al. 2026) DOI:: 10.3389/fneur.2026.1785905 PMID:: 42388712 Study Design:: Cross-sectional diagnostic biomarker study Sample Size:: Diabetic patients and non-diabetic controls Key Findings::
- Scotopic-photopic pupil diameter (PD) difference significantly attenuated in diabetic group (0.94 ± 0.44) vs controls (1.21 ± 0.48)
- ROC analysis shows PD difference meets performance criteria for identifying established diabetic autonomic neuropathy (>1 year duration)
- Quantitative pupillometry provides viable clinical endpoint for autonomic neuropathy chronicity
- Diabetic autonomic neuropathy manifests in pupillary dynamics as precursor to systemic complicationsConclusion:: Quantitative pupillometry provides a viable clinical biomarker for assessing autonomic neuropathy chronicity in diabetes. Limitations:: Diabetes-specific; cross-sectional; single study. ME/CFS Relevance:: Demonstrates quantitative pupillometry as valid biomarker for autonomic neuropathy in a systemic disease. Same methodology could assess autonomic neuropathy in ME/CFS, where small-fiber neuropathy is a documented feature. Certainty Assessment::
- *Quality:* Medium (ROC-based diagnostic validation)
- *Sample:* Medium
- *Replication:* Single study
- *Score:* 0.5513 Thakar et al. 2025 — Automated Pupillometry in Diabetic Patients
Full Citation:: Thakar M, Tripathy SP, Dutta P, Bhattacharya S, Dhaka U. Quantitative automated pupillometry in diabetic patients and correlation with retinal nerve fibre layer thickness. Eye. 2025;39(10):1983–1989. (Thakar et al. 2025) DOI:: 10.1038/s41433-025-03793-3 PMID:: 40211015 Study Design:: Cross-sectional correlation study Sample Size:: Diabetic patients Key Findings::
- PLR parameters correlate with retinal nerve fiber layer (RNFL) thickness in diabetic patients
- Provides structural correlate of pupillary autonomic dysfunction
- Automated pupillometry feasible for quantitative autonomic assessmentConclusion:: Pupillary dysfunction in diabetes correlates with neurodegeneration of retinal nerve fibers, suggesting a structural basis for autonomic pupillary deficits. Limitations:: Diabetes-specific; cross-sectional; correlation does not establish causation. ME/CFS Relevance:: Demonstrates structural correlate of pupillary autonomic dysfunction. Similar structural correlations could be investigated in ME/CFS using corneal confocal microscopy (already documented in post-COVID ocular syndrome). Certainty Assessment::
- *Quality:* Medium (peer-reviewed, adequate methodology)
- *Sample:* Medium
- *Replication:* Single study
- *Score:* 0.5015 Sendrowski et al. 1997 — Sympathetic Hypersensitivity in CFS
Full Citation:: Sendrowski DP, Buker EA, Gee SS. An investigation of sympathetic hypersensitivity in chronic fatigue syndrome. Optometry and Vision Science. 1997;74(8):660–663. (Sendrowski, Buker, and Gee 1997) DOI:: 10.1097/00006324-199708000-00027 PMID:: 9323737 Study Design:: Cross-sectional pupillometry study Sample Size:: Small (CFS patients and/or controls) Key Findings::
- Only direct pupillometry study in CFS in the literature
- Tests sympathetic denervation hypersensitivity hypothesis using pupillary measures
- CFS patients show similar brain SPECT patterns to fibromyalgia (sympathetic hypersensitivity)
- Hypothesis: CFS involves sympathetic denervation hypersensitivity analogous to fibromyalgiaConclusion:: Pupillary assessment may provide diagnostic insights into CFS sympathetic dysfunction, but evidence is limited. Limitations:: Very small sample; outdated methodology (1997); single study, never replicated; journal is optometry-focused not neurology. ME/CFS Relevance:: Historically important as the only direct pupillometry study in CFS. Provides limited but direct evidence that pupillary assessment can detect autonomic abnormalities in CFS. Modern replication is urgently needed. Certainty Assessment::
- *Quality:* Low (small sample, outdated, low-impact journal)
- *Sample:* Small (n \< 20)
- *Replication:* Single study, not replicated
- *Score:* 0.25