Autonomic Testing

Autonomic testing encompasses a battery of physiologic and neurochemical evaluations designed to assess the function of the sympathetic and parasympathetic nervous systems. Indications for autonomic testing include suspected autonomic neuropathy, orthostatic intolerance, small fiber neuropathy, postural orthostatic tachycardia syndrome (POTS), and differentiation of multiple system atrophy (MSA) from Parkinson disease. Because autonomic dysfunction may be subclinical for years before overt symptoms develop — particularly in diabetic cardiovascular autonomic neuropathy — standardized testing plays a critical role in early detection, localization of the lesion (preganglionic vs postganglionic), and monitoring disease progression. The Composite Autonomic Severity Score (CASS) integrates results from sudomotor, cardiovagal, and adrenergic domains into a single grading system that facilitates longitudinal tracking and comparison across autonomic disorders.

Cardiovascular Autonomic Reflex Tests (Ewing Battery)

Originally proposed by Ewing and colleagues in 1985, the Ewing battery consists of five noninvasive cardiovascular reflex tests that can be performed at the bedside or in an autonomic laboratory with an ECG and blood pressure cuff. Three tests assess parasympathetic (cardiovagal) function and two assess sympathetic (adrenergic) function. The American Diabetes Association recommends screening all patients with existing microvascular complications of diabetes for cardiovascular autonomic neuropathy using this battery.

Parasympathetic Tests

• Heart rate response to deep breathing (E:I ratio): The patient breathes at a controlled rate of 6 breaths per minute while continuous ECG records R-R intervals. The expiration-to-inspiration (E:I) ratio reflects the maximum and minimum heart rates during each respiratory cycle. Heart rate normally increases with inspiration and decreases with expiration via vagal modulation. A beat-to-beat variation of >15 beats per minute is normal; ≤10 beats per minute is abnormal. This is the most sensitive and reproducible test of cardiovagal function
• Valsalva ratio: The patient blows into a mouthpiece maintaining 40 mmHg expiratory pressure for 15 seconds. The Valsalva ratio is the maximum heart rate during strain (phase II) divided by the minimum heart rate after release (phase IV). A ratio ≥1.21 is normal; ≤1.10 is abnormal. The test evaluates both afferent baroreceptor pathways and efferent vagal output
• 30:15 ratio (lying-to-standing): The ratio of the longest R-R interval around the 30th beat after standing to the shortest R-R interval around the 15th beat. A ratio ≥1.04 is normal; ≤1.00 is abnormal. This immediate heart rate response to standing reflects the integrity of the cardiovagal baroreflex arc

Sympathetic Tests

• Blood pressure response to standing: Systolic and diastolic blood pressure are measured supine and after 3 minutes of standing. A sustained drop of ≥20 mmHg systolic or ≥10 mmHg diastolic defines orthostatic hypotension. This test evaluates sympathetic vasoconstrictor function
• Blood pressure response to sustained handgrip: The patient performs isometric handgrip at 30% of maximum voluntary contraction for up to 5 minutes. Diastolic blood pressure normally rises ≥16 mmHg; a rise <10 mmHg is abnormal. This test assesses sympathetic efferent pathways independent of baroreflex mechanisms

Interpretation and Staging

Tilt Table Testing

Head-up tilt table testing provides controlled orthostatic stress to evaluate cardiovascular autonomic regulation. The patient lies supine on a motorized table for a baseline period of 10–20 minutes with continuous heart rate and beat-to-beat blood pressure monitoring. The table is then passively tilted to 60–70° for up to 45 minutes while hemodynamic parameters are recorded. The test eliminates the confounding effects of skeletal muscle pumping that occur during active standing, providing a purer assessment of autonomic reflex function.

Hemodynamic Response Patterns

Sudomotor Function Testing

Sudomotor dysfunction is one of the earliest detectable neurophysiologic abnormalities in autonomic and small fiber neuropathies. Because sweat glands are innervated by postganglionic sympathetic cholinergic fibers, sudomotor testing provides a unique window into the integrity of sympathetic unmyelinated C fibers. Multiple complementary techniques exist, each with distinct strengths in localization and sensitivity.

Quantitative Sudomotor Axon Reflex Test (QSART)

QSART is the most widely used quantitative sudomotor test. Acetylcholine (10%) is iontophoresed into the skin at 2 mA for 5 minutes, stimulating local postganglionic sympathetic axon terminals. The axon reflex triggers release of acetylcholine from adjacent nerve terminals, activating nearby sweat glands. The evoked sweat response is recorded by a humidity sensor in an adjacent compartment over an additional 5 minutes. Standard testing sites include:

• Forearm: 75% of the distance from the ulnar epicondyle to the pisiform bone
• Proximal leg: 5 cm distal to the fibular head (lateral)
• Distal leg: 5 cm proximal to the medial malleolus (medial)
• Dorsal foot: Proximal dorsum of the foot

Thermoregulatory Sweat Test (TST)

The TST evaluates the entire sympathetic sudomotor pathway from the hypothalamus to the sweat gland. The patient’s skin is coated with an indicator powder (alizarin red-S, cornstarch, and sodium carbonate) that changes color from golden-orange to dark purple upon contact with sweat. The patient is placed in a controlled humidity chamber and core body temperature is raised by 1°C above baseline (or to 38°C) using infrared heating lamps. The resulting sweat pattern is photographed and analyzed.

• Global anhidrosis: Absent sweating over >80% of body surface area suggests diffuse autonomic failure or a preganglionic lesion
• Length-dependent pattern: Distal anhidrosis with preserved proximal sweating indicates a length-dependent neuropathy (eg, diabetic autonomic neuropathy, idiopathic small fiber neuropathy)
• Dermatomal or regional pattern: Non-length-dependent anhidrosis may indicate a central lesion, myelopathy, or focal autonomic neuropathy
• Combining TST with QSART: If TST shows anhidrosis and QSART is normal at the same site, the lesion is preganglionic; if both are abnormal, the lesion is postganglionic. This distinction is critical for differentiating MSA (preganglionic) from pure autonomic failure (postganglionic)

Other Sudomotor Tests

• Sudoscan (electrochemical skin conductance): Measures chloride-ion-dependent current flow through sweat glands on the hands and feet via stainless steel electrodes. Provides a rapid, noninvasive screening tool for sudomotor dysfunction with good sensitivity but limited ability to localize lesions
• Silastic sweat imprint: Pilocarpine iontophoresis stimulates sweat glands; the individual sweat droplet impressions are captured on a silastic mold. Allows quantification of both sweat volume and active sweat gland density, providing complementary information to QSART
• Sympathetic skin response (SSR): Records transient changes in skin electrical potential after a stimulus (deep inspiration, loud noise). Has high variability and limited sensitivity; generally considered a screening rather than a diagnostic test

Skin Punch Biopsy for Autonomic Nerves

Skin punch biopsy, traditionally used to quantify intraepidermal nerve fiber density (IENFD) for small fiber neuropathy diagnosis, can also assess autonomic innervation of dermal structures. A 3 mm punch biopsy taken to at least 4 mm depth is immunostained with PGP 9.5 (a pan-axonal marker) to visualize nerve fibers innervating sweat glands and arrector pili muscles.

Autonomic Nerve Fiber Assessments

• Sweat gland nerve fiber density (SGNFD): Quantifies sympathetic cholinergic fibers innervating eccrine sweat glands in the deep dermis. Reduced SGNFD correlates with impaired sudomotor function on QSART and may be more sensitive than QSART in detecting early autonomic small fiber loss
• Pilomotor nerve fiber density: Quantifies sympathetic adrenergic fibers innervating arrector pili muscles. Provides complementary assessment of a different autonomic fiber population and may detect abnormalities even when SGNFD is preserved
• Correlation with IENFD: SGNFD and IENFD often decline in parallel in length-dependent neuropathies, but dissociation can occur — isolated SGNFD reduction with normal IENFD suggests selective autonomic involvement
• Biopsy sites: Proximal thigh (20 cm below the anterior iliac spine) and distal leg (10 cm above the lateral malleolus) are standard; the foot can also be biopsied for sudomotor assessment
• Age- and sex-stratified normative values are available for SGNFD at multiple reference laboratories

Other Autonomic Tests

Plasma Catecholamines

Measurement of plasma norepinephrine levels in the supine position and after 5–10 minutes of standing is a valuable adjunct for differentiating causes of neurogenic orthostatic hypotension. Norepinephrine normally approximately doubles upon standing (typically from ~200 pg/mL supine to ~400 pg/mL standing). In postganglionic autonomic failure (eg, pure autonomic failure, diabetic autonomic neuropathy with postganglionic involvement), supine norepinephrine is low (<100 pg/mL) and fails to rise on standing. In preganglionic autonomic failure (eg, MSA), supine norepinephrine may be normal or low-normal but fails to increase appropriately (<60% rise) with orthostasis.

Urodynamic Studies

Cystometry and urodynamic evaluation assess parasympathetic (detrusor contraction) and sympathetic (internal sphincter tone) bladder function. Findings include detrusor underactivity, increased post-void residual volume, and impaired bladder sensation. Urodynamic abnormalities are common in diabetic autonomic neuropathy and MSA and may precede motor symptoms in MSA by years.

Gastric Emptying Scintigraphy

A standardized radiolabeled meal (typically egg substitute with toast and water) is ingested, and gamma camera imaging tracks gastric emptying over 4 hours. Retention of >60% at 2 hours or >10% at 4 hours confirms delayed gastric emptying (gastroparesis). This test evaluates vagal parasympathetic innervation to the stomach and is abnormal in up to 50% of patients with longstanding diabetes, though symptoms correlate poorly with objective delay.

Clinical Applications

Autonomic testing results must be interpreted within the clinical context. The following table summarizes expected findings across common autonomic disorders.

References

1. Ewing DJ, Martyn CN, Young RJ, Clarke BF. The value of cardiovascular autonomic function tests: 10 years experience in diabetes. Diabetes Care. 1985;8(5):491-498.
2. Low PA. Composite autonomic scoring scale for laboratory quantification of generalized autonomic failure. Mayo Clin Proc. 1993;68(8):748-752.
3. Freeman R. Autonomic peripheral neuropathy. Lancet. 2005;365(9466):1259-1270.
4. Low PA, Tomalia VA. Orthostatic hypotension: mechanisms, causes, management. J Clin Neurol. 2015;11(3):220-226.
5. Spallone V. Update on the impact, diagnosis and management of cardiovascular autonomic neuropathy in diabetes: what is defined, what is new, and what is unmet. Diabetes Metab J. 2019;43(1):3-30.
6. Pop-Busui R, Boulton AJM, Feldman EL, et al. Diabetic neuropathy: a position statement by the American Diabetes Association. Diabetes Care. 2017;40(1):136-154.
7. Chowdhury M, Nevitt S, Eleftheriadou A, et al. Cardiac autonomic neuropathy and risk of cardiovascular disease and mortality in type 1 and type 2 diabetes: a meta-analysis. BMJ Open Diabetes Res Care. 2021;9(2):e002480.
8. Novak P. Quantitative autonomic testing. J Vis Exp. 2011;(53):2502.
9. Low PA, Caskey PE, Tuck RR, Fealey RD, Dyck PJ. Quantitative sudomotor axon reflex test in normal and neuropathic subjects. Ann Neurol. 1983;14(5):573-580.
10. Fealey RD, Low PA, Thomas JE. Thermoregulatory sweating abnormalities in diabetes mellitus. Mayo Clin Proc. 1989;64(6):617-628.
11. Gibbons CH, Illigens BMW, Wang N, Freeman R. Quantification of sweat gland innervation: a clinical-pathologic correlation. Neurology. 2009;72(17):1479-1486.
12. Goldstein DS, Holmes C, Sharabi Y, Wu T. Cerebrospinal fluid biomarkers of central catecholamine deficiency in Parkinson disease and other synucleinopathies. Brain. 2012;135(pt 6):1900-1913.
13. Sheldon RS, Grubb BP II, Olshansky B, et al. 2015 Heart Rhythm Society expert consensus statement on the diagnosis and treatment of postural tachycardia syndrome, inappropriate sinus tachycardia, and vasovagal syncope. Heart Rhythm. 2015;12(6):e41-e63.
14. Vernino S, Low PA, Fealey RD, Stewart JD, Farrugia G, Lennon VA. Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies. N Engl J Med. 2000;343(12):847-855.
15. Kaufmann H, Norcliffe-Kaufmann L, Palma JA. Baroreflex dysfunction. N Engl J Med. 2020;382(2):163-178.
16. Elafros MA, Callaghan BC. Diabetic neuropathies. Continuum (Minneap Minn). 2024;30(5):1440-1472.
17. Lauria G, Hsieh ST, Johansson O, et al. European Federation of Neurological Societies/Peripheral Nerve Society guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Eur J Neurol. 2010;17(7):903-912.
18. Low PA, Benrud-Larson LM, Sletten DM, et al. Autonomic symptoms and diabetic neuropathy: a population-based study. Diabetes Care. 2004;27(12):2942-2947.
19. Gibbons CH, Freeman R. Treatment-induced neuropathy of diabetes: an acute, iatrogenic complication of diabetes. Brain. 2015;138(pt 1):43-52.
20. Palma JA, Kaufmann H. Autonomic disorders predicting Parkinson disease. Parkinsonism Relat Disord. 2014;20(suppl 1):S94-S98.