Handbook of Clinical Neurology, Vol.

160 (3rd series)

Clinical Neurophysiology: Basis and Technical Aspects
K.H. Levin and P. Chauvel, Editors
https://doi.org/10.1016/B978-0-444-64032-1.00028-X
Copyright © 2019 Elsevier B.V. All rights reserved

Chapter 28

Autonomic testing, methods and techniques

BEN M.W. ILLIGENS AND CHRISTOPHER H. GIBBONS*
Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States

Abstract
The evaluation of autonomic function requires indirect assessment of neurophysiologic function using
specialized equipment that is often available only at tertiary care centers, with few specialists available.
However, the evaluation of autonomic function is rooted in basic physiology, and the results can be inter-
preted by careful consideration of the context of the problem. Many automated devices have become
widely available to test autonomic function, but they tend to gather inadequate data leading to frequent
misdiagnosis and clinical confusion. We review the details necessary for the neurophysiologist to properly
perform, and interpret, autonomic function testing.

of autonomic function that occur across other organ sys-

INTRODUCTION
The autonomic nervous system (ANS) has diverse
end-organ innervation. As a consequence, the study of
autonomic function is expansive and typically targets
the particular end organ of interest. The result is a large
number of techniques to assess the function of the ANS
for both research and clinical purposes. These tests of
autonomic function assess the capacity of the system
to respond to a particular stimulus (Low, 2003; Low
and Benarroch, 2008). In contrast to tests of function,
gastric biopsies, sural nerve biopsies, or skin biopsies
to assess peripheral nerve fibers are structural tests of
the autonomic nervous system. However, structural
methods are invasive and limited by where biopsies
can be obtained, the invasive nature of the tests, and
the ability to repeat the tests. Therefore, functional tests
are generally preferred and have become the standard for
evaluation of the autonomic nervous system.
For neurologists, clinical autonomic testing focuses
on the assessment of the cardiovagal parasympathetic
function, the cardiovascular sympathetic adrenergic
function, and the sympathetic cholinergic sudomotor
systems (Low, 2003). There are many additional tests
tems, such as urodynamic testing in urology, gastric emp-
tying studies in gastroenterology, and pupillometry in
ophthalmology. Although these are important tests to con-
sider in the evaluation of autonomic end-organ function,
they are outside the scope of this chapter and the reader
is referred to several excellent resources for further infor-
mation (Smith and Smith, 1983; Gavriysky, 1995;
Sakakibara et al., 1997; Davies and Smith, 1999; Park
and Camilleri, 2006; Horvath et al., 2014).
Parasympathetic function is tested by measuring
changes in heart rate in response to deep breathing,
to the Valsalva maneuver, and active standing (Low,
2003). Sympathetic function is in general tested by mea-
suring changes in blood pressure in response to the
Valsalva maneuver, tilt table testing, and active standing.
Sudomotor function is usually measured with the
Quantitative Sudomotor Axon Reflex Test (QSART) or
the Thermoregulatory Sweat Test (TST) (Illigens and
Gibbons, 2009).
This chapter provides a general overview of clinical
autonomic testing, followed by a detailed description
of tilt table testing, autonomic reflex testing, and the
sudomotor tests QSART and TST.

*Correspondence to: Christopher H. Gibbons M.D. Center for Autonomic and Peripheral Nerve Disorders, Department of
Neurology, Beth Israel Deaconess Medical Center, 1 Deaconess Road, Boston, MA 02215, United States. Tel.: +1-617-632-
8454, Fax: +1-617-632-0852, E-mail: cgibbons@bidmc.harvard.edu
420 B.M.W. ILLIGENS AND C.H. GIBBONS
CLINICAL AUTONOMIC TESTING:
SYMPATHETIC ADRENERGIC AND
PARASYMPATHETIC
Autonomic testing is indicated for patients who have
a history of syncope, orthostatic hypotension, postural
tachycardia syndrome (POTS), thermoregulatory dys-
function, peripheral neuropathy, general autonomic
failure, or neurodegenerative disease. Symptoms of ortho-
static hypotension include postural lightheadedness, dizzi-
ness, weakness, coat hanger headache, or shortness of
breath (Gibbons and Freeman, 2005). Gastrointestinal
manifestations can include nausea, vomiting, diarrhea,
constipation, bloating or gastroparesis. Urologic mani-
festations may include urinary urgency, frequency
or retention. Sudomotor dysfunction may present
with anhidrosis, hyperhidrosis, or thermoregulatory
abnormalities.
By assessing cardiovagal parasympathetic function,
cardiovascular sympathetic adrenergic function, and
sympathetic cholinergic sudomotor function, the role
of the autonomic system in the pathophysiology of these
conditions and the contribution to these symptoms can be
evaluated (Low, 2003; Low et al., 2003).
In preparation for autonomic testing, patients should
stop medications (if it is safe to do so) that impact auto-
nomic function, for five half-lives. Practically, we gener-
ally recommend holding medications for 48 h before
testing, if this is possible. Test results may be influenced
by a number of classes of medications including those
with anticholinergic effects (such as tricyclic antidepres-
sants or serotonin–norepinephrine reuptake inhibitors),
antihistamines, decongestants, medications with anti-
muscarinic effects, medications that raise or lower blood
pressure, medications that influence volume status
(volume expanders such as fludrocortisone or volume
contractors such as furosemide), analgesics (opioids),
and antiinflammatory agents (Low, 2003, 2008). Some
exceptions are made for medications that could potenti-
ate medical complications, or create withdrawal effects if
removed (e.g., levothyroxine, opioids), or if the auto-
nomic testing is performed to assess the response on
medication or is repeated to assess the effect of a new
treatment (Low, 2003). On the day of testing, patients
should not use compression stockings and should avoid
caffeine and tobacco. Approximately 3 h before auto-
nomic testing, substantial fluid or food intake should
be avoided (Low, 2003).
During autonomic testing, the patient is equipped
with a brachial blood pressure cuff, a beat-to-beat blood
pressure monitor on the finger of the contralateral hand,
and a single- or multilead electrocardiogram (ECG). The
brachial blood pressure monitor is set to measure every
1–2 min during tilt table testing and active standing.
A beat-to-beat monitor and ECG measure continuously
throughout the duration of testing. Blood pressure and
heart rate are connected to a data acquisition system.
Patients are placed in supine position on a tilt test with
footrest support and stabilizing belts. After the patient
has assumed a relaxed, supine position a baseline ECG
is recorded to screen for any cardiac abnormalities
(e.g., arrhythmia, atrial-ventricular delay or block, long
PR interval, long QTc interval) that exist at baseline
and may warrant further investigation.
Heart rate variability to paced breathing
Deep breathing with maximum effort is performed for
nine cycles, with each cycle paced at 5 s for inhalation
and 5 s for exhalation to assess heart rate variability
(HRV) as a measure of parasympathetic function.
Normal HRV in response to deep breathing is an increase
of heart rate during inhalation and a decrease during
exhalation, also known as the respiratory sinus arrhyth-
mia (Angelone and Coulter, 1964; Davies and Neilson,
1967; Low, 2003). HRV data are analyzed and compared
to normative data adjusted for age and gender. Heart rate
variability declines with age. It is important to measure
respiratory effort in order to provide context to the
results. Data cannot be interpreted if the respiratory effort
is suboptimal. Automated devices that record heart rate
variability generally do not analyze respiratory effort
and therefore cannot appropriately interpret the results
(Gibbons CHC and Fife, 2012). A reduced HRV with
good effort may be a sign of isolated parasympathetic
dysfunction or may occur in the context of a generalized
dysfunction of the autonomic nervous system. Fig. 28.1
highlights several responses to paced breathing.
Valsalva maneuver
A test that measures both sympathetic adrenergic and
parasympathetic function is the Valsalva maneuver
(Sandroni et al., 1991; Low, 2003; Hilz and Dutsch,
2006), a forced breathing exercise with open glottis
and an expiratory pressure of 30–40 mmHg for 15 s
against resistance, which is used to assess the cardiovagal
heart rate response and the sympathetic adrenergic blood
pressure response (Low, 2003). The hemodynamic
changes during the Valsalva maneuver occur in a
sequence of four distinct phases (Gibbons and
Freeman, 2004). In phase I, the blood pressure increases
during the beginning of the expiratory effort, a pure
mechanical effect due to a sudden increase in intratho-
racic and intraabdominal pressure leading to compres-
sion of the aorta and vena cava. This is followed by
phase II, which is divided into early and late. During
early phase II the blood pressure decreases steadily
 AUTONOMIC FUNCTION TESTING 421

Fig. 28.1. Heart rate variability. The solid lines are the heart rate in response to paced breathing (the left Y-axis for A, B and C). The
dotted lines in the figure are the respiratory capacity (a higher number indicates a greater chest wall excursion and lung volume—
right Y-axis). In (A), there is normal heart rate variability that is time locked to deep respiration. In (B), there is significant respi-
ratory effort, but no change in heart rate (this is a patient with Parkinson’s disease and severe dysautonomia). In (C), the patient is
not making any effort at deep respiration with minimal heart rate variability. The heart rate response to breathing in (C) could easily
be mistaken for an autonomic neuropathy if respiratory effort were not monitored. Copyright B.M.W. Illigens.
422 B.M.W. ILLIGENS AND C.H. GIBBONS
secondary to the sustained compression of the vena cava,
leading to a decreased venous return to the heart, reduced
preload, reduced stroke volume, and therefore dimin-
ished cardiac output. The fall in blood pressure triggers
a compensatory heart rate increase from withdrawal of
cardiovagal tone. It also activates baroreceptors in the
carotid and aortic arch, causing an efferent sympathetic
discharge, which leads to increased total peripheral resis-
tance and ultimately a recovery of the blood pressure
marking late Phase II. Phase III is a sudden drop in blood
pressure due to a decrease in intrathoracic pressure once
the expiratory effort is terminated. Phase IV is the blood
pressure overshoot and depends on cardiac
adrenergic tone.
As a measure of parasympathetic function, the heart
rate response in the Valsalva maneuver is analyzed by
the Valsalva ratio (Low, 2003; Hilz and Dutsch, 2006).
The Valsalva ratio is a calculation of the maximum heart
rate during the maneuver over the minimum heart rate
during the 30 s after maneuver and is compared to norma-
tive data by age and gender. There is an age-related
decline in the Valsalva ratio. A larger Valsalva ratio Fig. 28.2. The phases of the Valsalva maneuver. The blood
indicates more “normal” parasympathetic function. The pressure (top tracing) and heart rate (middle tracing) in
assessment of heart rate variability during the Valsalva response to a Valsalva maneuver. The bottom tracing demon-
maneuver requires the blood pressure for appropriate strates the expiratory pressure. This is a normal response with
interpretation. If there is no drop in blood pressure during an appropriate dynamic change in blood pressure during
phase II because of inadequate respiratory effort, there phases 1–4, with an appropriate tachycardia in response to
will be no heart rate response. Automated devices typi- expiratory pressure of 40 mmHg for 15 s. Copyright B.M.W.
Illigens.
cally do not measure expiratory pressure or beat-to-beat
blood pressures, preventing appropriate interpretation
of the heart rate response to a Valsalva maneuver. Due to the increase in intrathoracic pressure that
A review of the phases of the Valsalva maneuver is pro- occurs during testing, a Valsalva maneuver is not recom-
vided in Fig. 28.2. mended for individuals who have undergone eye surgery
Sympathetic adrenergic function is assessed by the during the previous 3 months, or individuals with dia-
beat-to-beat blood pressure response during late phase betic retinopathy or untreated glaucoma.
II and during phase IV. A normal response would be a
recovery of the blood pressure within 4–7 s after phase
Active stand
I (defined as late phase II) and a blood pressure after
the Valsalva maneuver overshooting the baseline, An additional test of both parasympathetic and sympa-
defined as phase IV (Low, 2003; Hilz and Dutsch, thetic adrenergic function is the active stand (Gibbons
2006). Mild sympathetic dysfunction would be a reduced and Freeman, 2004). The active stand is performed after
and/or delayed recovery after 7 s in phase II and reduced at least 5 min baseline recording of blood pressure and
and/or delayed phase IV overshoot. Severe sympathetic heart rate in the supine, rested position. The patient
dysfunction would be a continued fall in blood pressure moves from the supine to the standing position quickly
throughout the expiratory effort with no evidence of (within 3 s if possible) and stands in an immobile, stable
a late phase II blood pressure recovery and absent over- position for 5 min. This test reflects the patient’s daily
shoot in phase IV (Sandroni et al., 1991). A technically experience in positional change. In response to standing
inadequate Valsalva maneuver is shown in Fig. 28.3. In there is a shift in blood volume of 300–800 mL from the
this example, values obtained could be interpreted as central intravascular compartment to the peripheral vas-
abnormal if the respiratory effort is not recorded. cular system (Ewing et al., 1980). With the sudden trans-
However, once the respiratory effort is considered in fer of blood volume there is a rapid increase in the heart
the analysis, it is clear that the test is not performed rate that peaks at about 3 s. This sudden, and transient,
properly. tachycardia is mediated by inhibition of vagal tone likely
 AUTONOMIC FUNCTION TESTING 423

Fig. 28.3. An inadequate Valsalva maneuver. The blood pressure (top tracing) and heart rate (middle tracing) in response to a
Valsalva maneuver. In this example, the patient does not generate sufficient respiratory effort to elicit a sufficient change in blood
pressure or heart rate. Without measurement of expiratory pressure and beat-to-beat blood pressure, it is not possible to differen-
tiate an abnormal response from inadequate effort. Copyright B.M.W. Illigens.

as a result of muscle contraction, referred to as the exer-
cise reflex. After the initial increase in heart rate, there is a
further baroreflex mediated increase in heart rate from 3
to 12 s in response to a fall in blood pressure due to dimin-
ished vasoconstrictor tone. The transient decrease in
blood pressure and increase in heart rate results in a
new baseline after approximately 30 s. As blood pressure
returns toward normal, there is a transient blood pressure
overshoot period and a baroreflex mediated bradycardia
at 30 s after initiation of standing (Ewing et al., 1980).
Sympathetic adrenergic function is measured by
changes in blood pressure while standing compared to
the baseline, supine position. A diagnosis of orthostatic
hypotension is defined as a 20-mmHg decrease in sys-
tolic, or a 10-mmHg decrease in diastolic, blood pressure
within 3 min of standing (Freeman et al., 2011). In
patients with supine hypertension, the fall in blood
pressure for a diagnosis of orthostatic hypotension is
30/15 mmHg (Freeman et al., 2011). During active stand-
ing, parasympathetic function is measured using the
30:15 ratio (Low, 2003). The 30:15 ratio is determined
by the slowest heart rate (longest RR interval) occurring
about 30 s after standing divided by the fastest heart rate
(shortest RR interval) occurring about 15 s after standing.
The larger the value for the 30:15 ratio, the more “intact”
the parasympathetic nervous system is. Abnormalities
that can be detected on active standing include evidence
of parasympathetic dysfunction, orthostatic hypoten-
sion, or postural tachycardia. The typical parasympa-
thetic function (heart rate) response to an active stand
is shown in Fig. 28.4.
The only contraindication against standing is a phys-
ical disability that would prevent the ability to stand
safely. In patients with severe orthostatic hypotension
and in those who had neurally mediated syncope during
tilt table testing, care should be taken to perform the test
424 B.M.W. ILLIGENS AND C.H. GIBBONS

Fig. 28.4. The 30:15 ratio. The 30:15 ratio is a quantified change in the heart rate that occurs between the 15th and 30th second, or
heartbeat, after standing, during which time a significant change in heart rate occurs. This figure demonstrates the heart rate
response to moving from a supine to standing position. The R–R interval at the lowest heart rate (generally around 30 s, or
30th heart beat) is divided by the R–R interval of the highest heart rate (typically at 15 s, or the 15th heart beat) to provide a numer-
ical measure of parasympathetic activity. The exact peak and trough of heart rate will vary between individuals and will be related
to the underlying heart rate; therefore the 30:15 ratio can refer to either the number of heartbeats after standing or the time in
seconds and is just a rough estimate of where to select the maximum and minimum heart rates. Copyright B.M.W. Illigens.

in a safe environment where individuals can be protected
from a fall. It should be noted that the results must be
interpreted within the clinical context for the individual.
Orthostatic hypotension can be a consequence of medi-
cation use, volume depletion, or other medical condition
and does not necessarily indicate a problem with the
autonomic nervous system.
Tilt table testing
The tilt table test is used to assess the hemodynamic
response to postural change. Postural change from the
supine to the upright position leads to venous pooling,
a process where 500–1000 mL blood shifts from the tho-
rax into the legs and to a lesser extent into the abdominal
and pelvic regions (Wieling et al., 1985; Ajsmit
et al., 1996; Low, 2003). Several countermechanisms
exist, but the most important are the neurocardiovascular
system and the muscle-venous pump. During the active
stand both mechanisms are utilized to maintain blood
pressure. During the tilt table test, otherwise known as
a “passive stand,” the individual is brought to a 60- to
80-degree tilt, a position where leg muscles are used to
a minimum because the individual is supported against
a bed (Wieling et al., 1985; Ajsmit et al., 1996).
During the tilt table test, both the parasympathetic and
the sympathetic adrenergic systems are tested through
gravitational stress. Normally, upon tilt up there is a
transient blood pressure drop due to venous pooling,
which then triggers the baroreceptors in the carotid artery
and aortic arch (Wieling et al., 1985; Sprangers et al.,
1991). The baroreceptors then cause a compensatory
heart rate increase through withdrawal of parasympa-
thetic tone and an increase in blood pressure due to an
increase in sympathetic adrenergic tone. The following
conditions can be identified during tilt table testing:
neurally mediated syncope, orthostatic hypotension,
POTS, and delayed orthostatic hypotension (Freeman
et al., 2011).
The patient will start the tilt test in the supine position
on a table equipped with a footrest support and suspen-
sion belts to ensure a secure and safe testing environment
during the tilt. The patient should rest in a recumbent
position for a prolonged period of time, generally about
20 min. After at least three similar baseline blood pres-
sure and heart rate readings are acquired, the patient is
quickly tilted head-up to 60–80 degrees (the exact angle
varies across different labs, but we prefer a 60-degree
angle). The patient is instructed to avoid moving the legs
during the test (to avoid using the muscle pump) and is
then observed for symptoms and hemodynamic changes.
The patient is kept in the tilted position for at least 10 min
or until a terminating event occurs. Termination of the tilt
is based on clinical judgment, but the typical reasons for
tilting down a patient are (1) reduced responsiveness or
unresponsiveness (the clinician must use judgment; if
 AUTONOMIC FUNCTION TESTING 425

Fig. 28.5. Tilt table testing. This figure highlights four typical responses seen during tilt table testing. The thick black line is the heart
rate (left Y-axis) while the thinner black line is the beat-to-beat blood pressure (right Y-axis). In (A), there is a normal blood pressure
and heart rate response to tilt. In (B), there is a significant increase in heart rate in response to tilt with no change in blood pressure
(a postural tachycardia). In (C), there is a sudden hypotensive and bradycardic event seen at 18 min consistent with neurally mediated
syncope. In (D), there is a slow but progressive decline in blood pressure consistent with orthostatic hypotension. There is no
significant change in heart rate with the fall in blood pressure consistent with autonomic failure. Copyright B.M.W. Illigens.

there is no hemodynamic change this could represent
“pseudo-syncope”), (2) extreme discomfort and request
for early termination of testing, (3) sustained hypoten-
sion (this will depend on symptoms and change from
baseline blood pressure), (4) hypotensive bradycardia
consistent with neurally mediated syncope.
Many centers limit the duration of the tilt to 10 min,
but a duration of 45 min should be considered to rule
out delayed orthostatic hypotension and neurally medi-
ated syncope (Gibbons and Freeman, 2006). Both
delayed orthostatic hypotension and neurally mediated
syncope are often detected in the latter part of testing.
After tilt-down, another 3 or more minutes of recovery
are recorded. In the setting of profound hypotension or
neurally mediated syncope, a prolonged recovery time
may be required, often in the reverse Trendelenburg
position for a few minutes before returning to the supine
position. Examples of disorders seen on tilt table testing
are shown in Fig. 28.5.
A normal hemodynamic response is a systolic blood
pressure decrease of less than 20 mmHg, a diastolic pres-
sure decrease of less than 10 mmHg, and a heart rate
increase of not more than 30 beats per minute (these values
are for adults; children will vary by age) (Freeman
et al., 2011).
There are no contraindications to tilt table testing with
the exception of weight that exceeds the capacity of the
table. Each tilt table is rated to a maximum weight, and all
patients should be weighed to ensure that they do not
exceed the maximum capacity of the table. During tilt
table testing, there is a potential for a bradycardic, hypo-
tensive response that can be profound in some patients
with neurally mediated syncope (also known as vasova-
gal syncope). A code cart should be available in close
proximity and testing supervised by a physician with
appropriate training. In the setting of neurally mediated
syncope, the patient should immediately be placed in
the reverse Trendelenburg position. With prolonged
426 B.M.W. ILLIGENS AND C.H. GIBBONS
asystole an emergency alert should be called. In our
experience with thousands of tests, even prolonged
asystole recovers to normal sinus rhythm within 60 s
of reverse Trendelenburg. If unsupervised patients are
left in the head-up tilted position, this can result in dan-
gerously prolonged asystole (Maloney et al., 1988;
Milstein et al., 1989; Dangovian et al., 1992).

Other tests of sympathetic

adrenergic function
Several tests of autonomic function have been used clin-
ically, but not routinely, in the evaluation of autonomic
function. These tests include blood pressure response
to sustained handgrip, blood pressure response to mental
stress, and blood pressure response to cold water immer-
sion (the cold pressor test) (Ewing et al., 1976; Hague
et al., 1978; Wood et al., 1984; Vita et al., 1986;
Hjemdahl et al., 1989; Specchia et al., 1991). These tests
have been used for research purposes, and reported in
some clinical cases, but are not routinely used in clinical
practice because of the variability of response across
individuals, and lower sensitivity and specificity.
CLINICAL AUTONOMIC TESTING:
SUDOMOTOR (SYMPATHETIC
CHOLINERGIC) FUNCTION
Intact sudomotor function is important for thermo-
regulation and is mediated via eccrine sweat glands that
underlie sympathetic control. Thermoregulatory activity
originates in the hypothalamus and is mediated via pre-
ganglionic cholinergic neurons that synapse in the para-
vertebral ganglia with postganglionic neurons (Illigens
and Gibbons, 2009). The postganglionic axons extend
to the eccrine sweat glands and release acetylcholine
from the axon terminals into the junctional space where
the acetylcholine binds to postjunctional M3 muscarinic
receptors on eccrine sweat glands to provoke sweat
production.
The sudomotor axon reflex can be provoked by
cholinergic agents, such as acetylcholine, pilocarpine,
or methacholine (Low et al., 1983; Maselli et al., 1989;
Low et al., 1990). Upon stimulation, a direct sweat
response is elicited when the cholinergic agent binds to
M3 muscarinic receptors on sweat glands. At the same
time, the cholinergic agent binds on prejunctional nico-
tinic receptors on sudomotor axon terminals and gener-
ates an axon potential. In the case of the sudomotor
axon reflex, the action potential initially travels anti-
dromically along the postganglionic sympathetic sudo-
motor axon until it reaches a branch point. After the
branch point the potential spreads and travels orthodro-
mically along other axon branches into adjacent areas
of the skin, where neighboring populations of eccrine
Fig. 28.6. The sudomotor axon reflex. An example of the
sudomotor axon reflex. As acetylcholine is iontophoresed into
the skin, it activates a local nerve terminal and a signal is
sent antidromically up the nerve fiber. At a branch point, the
signal continues to propagate antidromically but also spreads
orthodromically down to neighboring sweat glands. These
neighboring glands also produce sweat outside the area of stim-
ulation (the indirect sweat response). Copyright B.M.W.
Illigens.
sweat glands are stimulated to produce sweat outside
of the area of direct stimulation (indirect axon-reflex
mediated sweat response) (Fig. 28.6) (Low et al.,
1983). A number of tests of sudomotor function have
been developed, including the acetylcholine sweat-spot
testing, silicone impressions, and the quantitative direct
and indirect axon reflex testing (Stewart et al., 1994;
Gibbons et al., 2008; Illigens and Gibbons, 2009). The
most widely used, and well established for clinical
purposes, is the QSART. It should be noted that many
autonomic sudomotor reflex tests use iontophoresis of
a cholinergic agent and measure sweat function in an
“axon reflex mediated” adjacent area.
Quantitative sudomotor axon reflex test
The QSART is a sudomotor axon reflex based method
to evaluate sympathetic cholinergic function. Axon
mediated sweat production is stimulated through ionto-
phoresis of a cholinergic agent and is measured in the
axon-reflex area on the skin with a multicompartment
capsule that is connected to a hygrometer to quantify
changes in humidity (Low et al., 1983).
Testing requires placement of a special multicompart-
mental sweat capsule with an outer ring compartment for
stimulation and an inner circular compartment for
recording on the skin. Standard recording sites are the
ventral forearm, lateral proximal leg, medial distal
leg, and dorsum of the foot. The outer ring of the capsule
 AUTONOMIC FUNCTION TESTING
is filled with a cholinergic agent of choice (typically 10%
acetylcholine in water). The inner compartment’s inflow
is connected to a nitrogen gas tank (or dehumidified air)
and the compartment’s outflow to a hygrometer. The
dehumidified gas is delivered at a constant flow rate to
the recording compartment located over the indirect area.
The recording typically shows an initially high level of
humidity (from room air still being in the system) that
slowly decreases until stabilizing at a low baseline level.
Iontophoresis of the cholinergic agent is then started at
2 mA for 5 min. Any change in humidity of the outflow
air during stimulation and for an additional 15 min of
recovery is recorded. Data are analyzed by determining
sweat onset latency, peak sweat production, and area
under the curve as a measure of sweat volume.
In a normal response, humidity increases from base-
line levels with a delay of 1–2 min after the initiation
of iontophoresis triggers sweat production (the delay is
due to the axon-reflex dependent delay in sweat produc-
tion in the indirect area and the time needed for the air to
travel from the recording compartment to the hygrome-
ter) and then steadily increases during and for up to
5 min after stimulation, followed by a slow decrease dur-
ing recovery. Abnormal responses are an increased,
decreased, or absent sweat response or a delay (latency)
until an increase in humidity is recorded. A length-
dependent neuropathy (such as in diabetes) would show
a stronger decrease in sweat volume in the distal record-
ing sites and milder or no decrease in more proximal
recording sites (Kennedy et al., 1984; Gibbons et al.,
2013). QSART testing is demonstrated in Fig. 28.7.
There are a number of potential limitations to QSART.
QSART can be expensive and the iontophoresis of acetyl-
choline to induce sweating can create confusion with phar-
macy staff who are not familiar with this technique. It is
important to control for ambient temperature and humidity
during testing. Results need to be interpreted based on
both age and gender. Medication use, topical applications
of moisturizers, and hydration status can contribute to spu-
rious results (Illigens and Gibbons, 2009). There are few
side effects from testing; some patients may notice some
skin irritation at the site of iontophoresis.
Thermoregulatory sweat testing
The TST measures the integrity of both the central and
peripheral sudomotor system from the brain to the sweat
glands (Illigens and Gibbons, 2009). The patient is
placed in a chamber that raises the core body tempera-
ture, producing a topographical distribution of sweat pro-
duction. For the TST, the patient is asked to wear only
underwear and is positioned supine on a table in a
temperature- and humidity-controlled room or chamber.
The temperature is set to 45–50°C and relative humidity
427
to 35%–40%. Wearing a facemask to protect the airway,
the patient is covered with an indicator dye that changes
color upon contact with moisture, enabling visualization
of the degree and distribution of sweat production (spar-
ing the area around the eyes, but including cheeks and
forehead). Commonly used indicators are alizarin red
powder (applied as a mixture of alizarin red with corn
starch and sodium carbonate (1:2:1)) and iodine–
cornstarch (iodine solution applied onto the skin and
dried and then covered by a thin layer of cornstarch).
In addition to creating a messy environment, these sub-
stances may also be a challenge to pharmacy staff who
are not familiar with their use. Areas of sweating will
cause a change in the indicator’s color (from yellow to
purple if alizarin red is used and from white to blue if
starch with iodine is used) and digital photographs are
taken to quantify the percentage of anterior body surface
area positive for sweat (expressed as TST% ¼ area of
anterior body anhidrosis divided by total body surface
area, multiplied by 100) (Low et al., 2006; Illigens and
Gibbons, 2009). The patient is equipped with oral and
skin temperature probes to measure core and body sur-
face temperature. Using temperature-regulated infrared
heaters positioned over the patient, the aim is to raise
the patient’s core temperature by at least 1.0°C above
baseline temperature or to 38°C (whichever is higher)
while maintaining a skin temperature of 38.5–39.5°C
to prevent overheating. A representative maximal sweat
response should be evoked within 30–65 min and total
testing time should not exceed 70 min to avoid hyperther-
mia (Illigens and Gibbons, 2009).
A normal sweat response shows a symmetric distribu-
tion pattern of sweat production over the entire body sur-
face at varying degrees of intensity. An abnormal sweat
response may present with an asymmetric sweat pattern
or with areas of anhidrosis that may be focal, segmental,
regional, or length-dependent (Illigens and Gibbons,
2009). To differentiate whether areas of sudomotor
dysfunction localized with the TST are due to lesions
in the preganglionic or postganglionic neuron, the
TST is combined with a test of postganglionic function,
such as QSART; a preganglionic lesion will demonstrate
an abnormal pattern of sweating with a normal QSART
response. A postganglionic lesion will demonstrate
abnormalities in both tests (Low, 2003). Examples of
thermoregulatory sweat testing are shown in Fig. 28.8.
The TST is a time-consuming and labor-intensive
test. It requires dedicated space, specialized heating
equipment, and appropriate shower facilities for patients
after testing. It is not considered a routine test in many
autonomic testing centers. Close observation of patient
status is required during testing; individuals with signif-
icant anhidrosis are at substantially increased risk of
hyperthermia.
428 B.M.W. ILLIGENS AND C.H. GIBBONS

Fig. 28.7. Quantitative sudomotor axon reflex testing (QSART). An example of the QSART set-up is provided. An iontophoretic
device provides the electrical current that allows acetylcholine to penetrate into the skin. The sweat capsule blows nitrogen gas
(QSART) or dehumidified air (QSWEAT) across the skin and a hygrometer measures the change in humidity. A sample sweat
output response is provided at the top of the display. Three sites on the leg and one site on the forearm are typically tested. Copyright
B.M.W. Illigens.

RESEARCH TESTING FOR A number of these functional autonomic tests are

AUTONOMIC FUNCTION
Cutaneous autonomic reflex tests
In addition to clinical autonomic tests, a number of tests
are used for research purposes to investigate autonomic
function. Testing the autonomic nervous system is fre-
quently performed through indirect functional tests to
assess the peripheral portion of the autonomic nervous
system. The skin is easily accessible and there are options
to test in multiple locations to evaluate contralateral or
length-dependent differences. Dysfunction of the periph-
eral autonomic nervous system may be the earliest mani-
festation of a small fiber neuropathy (Low et al., 2006).
based on the quantification of the autonomic axon reflex.
The term axon reflex was first used by Langley for an
axon-mediated pilomotor response (Langley, 1903).
Currently, the term axon reflex is most commonly asso-
ciated with Sir Thomas Lewis, who described the vaso-
motor axon reflex as the underlying mechanism for the
flare component of the triple response (Lewis, 1927).
In fact, the axon reflex exists for all three components
of the peripheral autonomic nervous system as a vaso-
motor response, a pilomotor response, and a sudomotor
response – and for all of them a quantitative approach
exists (Gibbons et al., 2008; Siepmann et al., 2012;
Illigens et al., 2013).
 AUTONOMIC FUNCTION TESTING 429

Fig. 28.8. Thermoregulatory sweat testing (TST). In response to a rise in core temperature, the body produces sweat for thermo-
regulatory purposes. With application of an indicator dye, a topographical map of sweat production can be measured. (A) A normal
sweat pattern in response to a rise in core temperature; (B) a pattern due to length dependent polyneuropathy; (C) a spinal cord
injury prevents sweat production below the level of the injury; (D) patterns of a right lateral femoral cutaneous nerve injury and a
right T10 radiculopathy; (E) global anhidrosis is detected. Copyright B.M.W. Illigens.

The vasomotor axon reflex is based on activation of
cutaneous afferent polymodal nociceptor C-fibers and
can be elicited by chemical stimuli, such as acetylcho-
line, and physical stimuli. Upon activation of those
fibers, an action potential is generated, which causes
local release of vasodilating neuropeptides and sweat
production in this area (direct vasomotor response), but
also travels orthodromically along those afferent fibers
until it reaches a branch point (Stewart et al., 1992;
Peltier et al., 2009; Illigens et al., 2013). The action
potential then spreads antidromically along axon
branches into neighboring areas of the skin until it
reaches the nerve terminals, where it triggers a release
of vasodilating neuropeptides with changes to vascular
flow outside of the initial area of activation (indirect
vasomotor response) (Low et al., 1983). The most com-
mon tests of vasomotor function are laser Doppler flow-
metry (LDF) and laser Doppler imaging (LDI), typically
measured in response to iontophoresis of acetylcholine
(Illigens et al., 2013). LDF measures changes in capillary
blood flow using a single point (or multipoint) laser. With
LDF, several single-point probes are utilized simulta-
neously to allow quantification of both direct and
axon-mediated changes in vasomotor response. LDI is
a technique that measures changes in capillary flow
by repeated scanning of a defined area of the skin with
a single point moving laser and has thus good two-
dimensional spatial and temporal resolution (Green
et al., 2009; Illigens et al., 2013). LDI does not measure
the same point over time like LDF; instead it uses a single
laser to create a topographical map. A normal vasomotor
response leads to very quick vasodilation in the direct
area measured by an increased capillary blood flow, fol-
lowed shortly by vasodilation in the indirect area. The
vasodilation peaks within 5 min and then slowly returns
to baseline several minutes after that. A reduction in
vasodilation, a delay in peak vasodilation, and/or a
decrease in the area of indirectly stimulated axon reflex
mediated vasodilation may be signs of peripheral nerve
dysfunction. However, spatial resolution of LDF is low
and measurements show high intra- and interindividual
variability, reducing the sensitivity and specificity of this
technique for individual patients. LDI is limited by a rather
complex data analysis and is also only useful for detecting
differences between groups of subjects. Both LDI and
LDF require expensive hardware, have a complex set-
up, and due to their limitations are currently confined to
the research setting (Green et al., 2009; Illigens et al.,
2013). At this time, we do not recommend these tests
for clinical evaluation of cutaneous autonomic vasomotor
function. Examples of these tests are shown in Fig. 28.9.
The pilomotor axon reflex can be elicited by mechan-
ical, thermal, electrical, or pharmacological stimuli, such
as phenylephrine, upon which C-fibers are activated to
produce a direct goose bump reaction and via axon reflex
an indirect goose bump reaction outside of the area of
direct stimulation (Siepmann et al., 2012).
A quantitative pilomotor axon reflex test has recently
been developed to assess this response (Siepmann
et al., 2012). The number of goose bumps, volume of
goose bumps, and area of spread of the indirectly acti-
vated goose bumps can be assessed. This technique is
currently used for research studies and is not recom-
mended for clinical use at this time.
430 B.M.W. ILLIGENS AND C.H. GIBBONS

Fig. 28.9. Laser Doppler flowmetry and imaging. The top images demonstrate the laser Doppler imaging (LDI) seen in response to
stimulation with acetylcholine (the brighter the color, the greater the blood flow). This provides a graphical representation of blood
flow over a region of skin. In the lower image the blood flow is measured by laser Doppler flowmetry (LDF) in response to ace-
tylcholine iontophoresis. LDI measures blood flow continuously from several discrete sites, while LDF measures blood flow over
an area at predetermined time points. Copyright B.M.W. Illigens.

Microneurography
Microneurography is the only test to directly evaluate
autonomic function (Wallin and Sundlof, 1979). Devel-
oped in the 1960s, microneurography measures the post-
ganglionic discharges in sympathetic axons using a
microelectrode. Sympathetic nerve fibers discharge
spontaneously in irregular bursts, but the activity
increases in response to orthostatic stress or with changes
in blood pressure (Charkoudian et al., 2005). The most
common site selected for microneurography studies is
the peroneal nerve at the fibular head; however, other
nerve fascicles can be studied as well. In studies of motor
nerve fascicles (such as the peroneal nerve), the multiunit
bursts contain primarily vasoconstrictor information and
are in synchrony with the cardiac rhythm. In skin fasci-
cles, the sympathetic bursts may contain information
about vasoconstriction, vasodilation, or sudomotor func-
tion, and the bursts are not typically linked to the cardiac
rhythm (Macefield et al., 2002). Microneurography
plays a very important role in research investigation of
autonomic function, but requires highly specialized
training, is very labor intensive, and is therefore is not
used for clinical purposes. An example of microneuro-
graphy is shown in Fig. 28.10.
 AUTONOMIC FUNCTION TESTING 431

Fig. 28.10. Microneurography. The upper image demonstrates a microneurography needle being placed in the common peroneal
nerve at the fibular head to measure muscle sympathetic nerve activity. In the lower figure, the heart rate (upper tracing, measured
by RR interval), the blood pressure (middle tracing) and muscle sympathetic nerve activity (lower tracing) can be seen. In response
to nitroprusside, there is a decrease in blood pressure with an increase in heat rate. Muscle sympathetic nerve traffic increases
during the drop in blood pressure. When blood pressure returns to normal (after administration of phenylephrine) the muscle sym-
pathetic nerve activity decreases. Copyright B.M.W. Illigens.
432 B.M.W. ILLIGENS AND C.H. GIBBONS
SUMMARY
The evaluation of autonomic function is an important
part of the neurophysiologic evaluation of disease. These
tests of autonomic neurophysiology extend, but do not
replace, the clinical evaluation of patients with suspected
autonomic disorders. The purpose of these tests is to pro-
vide objective evidence of autonomic function to assist
in diagnosing or managing disease. A battery of tests
is typically employed to characterize the parasympa-
thetic, sympathetic adrenergic, and sympathetic cholin-
ergic systems. Additional autonomic evaluation of
other end-organ systems may be considered through
collaboration with gastroenterology, cardiology, urol-
ogy, or ophthalmology. Special considerations include
testing space requirements, the sophisticated instruments
needed, costs of testing, interfering medications, and
hydration status, patient cooperation, and coexisting
medical conditions that either limit reliability of the
results or pose health risks during testing. Despite these
challenges, autonomic function testing is a valuable tool
in the diagnosis and management of neurologic disease.
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