Introduction
The dynamic interplay between the brain and the heart, mediated primarily through the autonomic nervous system (ANS), provides essential insights into cardiovascular (CV) regulation and overall physiological functioning of the vascular system. The ANS, comprising the sympathetic and parasympathetic branches, plays a central role in regulating heart rate (HR), vascular tone, and myocardial contractility. (1) Through continuous modulation of these functions, the ANS maintains CV homeostasis in response to both internal and external stimuli. Cardiac autonomic regulation involves complex reflex mechanisms, including baroreceptor and cardiopulmonary reflexes, as well as direct innervation of the heart by vagal and sympathetic fibers. (2,3)
Disruption of this delicate balance is often characterized by excessive sympathetic activation and/or parasympathetic withdrawal and has been implicated in the pathogenesis of various CV diseases, (4) including hypertension (HTN), (5) heart failure (HF), (6) arrhythmias, (7) and ischemic heart disease. (8) Autonomic dysfunction not only contributes to disease onset and progression but is also associated with adverse clinical outcomes, including increased morbidity and mortality. (9) As such, the assessment of autonomic function holds substantial clinical relevance for early detection, risk stratification, therapeutic decision-making, and prognostic evaluation in CV medicine. (9,10) Despite its widely-recognized importance, standardized autonomic testing remains underutilized in routine cardiology practice, largely due to limited awareness, inadequate training among clinicians, and constrained availability of testing infrastructure and validated protocols.
A variety of non-invasive tests are available for the assessment of ANS function, including heart rate variability (HRV), baroreflex sensitivity (BRS), blood pressure (BP) responses to physiological stimuli, and tilt-table testing. (10) Advanced methods such as microneurography and skin sympathetic nerve activity provide direct or surrogate measures of sympathetic outflow, while cardiovagal and sympathetic reflex tests (e.g., deep breathing, Valsalva maneuver) remain essential for functional evaluation. Novel indices such as HR acceleration and deceleration capacity, as well as complementary vascular measures such as pulse wave velocity and arterial stiffness, offer additional insights into autonomic regulation, particularly for subclinical or early-stage dysfunction. (10-12) These diagnostic tools can help clinicians detect subtle forms of dysautonomia, monitor the efficacy of therapeutic interventions, and improve individualized patient management. (12)
The aim of this article is to provide a comprehensive overview of ANS testing, with a particular focus on its application in cardiology. We will present the physiological foundations, methodological principles, clinical utility, and interpretative frameworks of each test. By synthesizing current knowledge and emphasizing the potential of these tools, we aim to encourage their broader integration into CV diagnostics and patient care.
HEART RATE VARIABILITY
Heart rate variability (HRV) refers to the physiological phenomenon of variation in the time intervals between consecutive heartbeats, typically measured as the time between successive R-waves (RR intervals) on an electrocardiogram (ECG). (13) This variability does not reflect pathological irregularity but rather the heart’s physiological adaptability to changing internal and external conditions and is considered a key non-invasive marker of ANS function. (13,14) HRV arises from the continuous and dynamic interaction between the sympathetic and parasympathetic divisions of the ANS in regulating sinoatrial node activity (SNA). Higher HRV generally indicates greater vagal (parasympathetic) influence and better CV adaptability, whereas reduced HRV is often associated with autonomic dysfunction, increased sympathetic dominance, and heightened risk of adverse CV outcomes. (15) As such, HRV provides valuable insight into the balance of autonomic control, stress reactivity, recovery capacity, and overall CV health. In CV pathology, an imbalance between sympathetic and parasympathetic activity may contribute to disease progression and poor clinical outcomes. (15,16) Therefore, HRV analysis serves not only as a diagnostic tool but also as a prognostic marker in various cardiac conditions. (16)
The quantitative assessment of HRV is facilitated through advanced, validated software platforms capable of extracting a comprehensive range of HRV indices from ECG recordings (Figure 1). (17) These parameters can be derived from various types of ECG data, including short-term recordings (typically 5 minutes in duration), (17) continuous 24-hour Holter monitoring, (18,19) as well as ECG segments obtained during or in the recovery phase after standardized exercise testing protocols. (20) Among available modalities, 24-hour Holter ECG recording remains the most diagnostically robust and clinically informative approach to HRV assessment, owing to its superior predictive validity in CV risk stratification and its capacity to support comprehensive analyses, including the time-domain, frequency-domain, and non-linear metrics. (19,21)
For example, 24-hour and short-term HRV recordings have both shown prognostic value in patients with HF, but long-term Holter recordings provide clearer differentiation among NYHA functional classes. (21) Additionally, HRV measurements derived from 24-hour recordings correlate significantly with BRS metrics in studies of healthy subjects, demonstrating that long-term HRV captures autonomic regulatory features that complement direct BRS assessment. (22) Crucially, HRV parameters can be extracted from standard ECG data, thereby facilitating their integration into routine clinical workflows and enabling longitudinal patient monitoring. These indices provide quantitative markers of autonomic balance and cardiac autonomic regulation under real-world physiological conditions. The most commonly assessed HRV parameters are summarized inTable 1. (13,23)
Regarding clinical interpretation and prognostic relevance of HRV in cardiac patients, substantial evidence supports HRV as a clinically useful, non-invasive biomarker of ANS dysfunction across a broad spectrum of CV conditions, including myocardial infarction (MI), HF, arrhythmias, and sudden cardiac death (SCD). This is strongly supported by large-scale systematic reviews and meta-analyses, which provide robust evidence for the association between reduced HRV and increased mortality risk in both healthy individuals and patient populations. (24) More recent observational and mechanistic studies further suggest that HRV analysis may facilitate early identification of autonomic dysregulation, improved phenotyping of patient with hypertension, and monitoring of therapeutic response, especially in hypertensive subtypes characterized by heightened sympathetic activity. (25) While these findings are largely derived from cross-sectional and cohort-based designs, they provide clinically relevant insights into autonomic patterns across different hypertensive syndromes.
In patients recovering from MI, prospective cohort studies using standardized short-term Holter recordings have demonstrated that markedly reduced HRV parameters – particularly low SDNN and elevated LF/HF ratio – are associated with an increased risk of arrhythmic events and adverse cardiac outcomes, even in patients with preserved ejection fraction. (26) Recent single-center cohort studies indicate that higher parasympathetic markers (RSMSSD and HF components) and a lower LF/HF ratio are independently associated with a reduced risk of atrial fibrillation (AF) following catheter ablation. These findings suggest that HRV parameters may hold independent prognostic value, complementing conventional diagnostic and risk stratification tools in clinical cardiology. (27)
Despite its well-established prognostic relevance, the diagnostic utility of HRV as a standalone parameter remains limited due to its susceptibility to numerous confounding factors. These include age, circadian rhythms, physical conditioning, metabolic status, pharmacological therapy (such as β-blockers, ACE inhibitors), respiratory patterns, and psychological or emotional states, all of which can significantly influence HRV measurements and reduce their interpretability. Therefore, although reduced HRV represents a sensitive marker of autonomic dysregulation, it lacks disease specificity. Its diagnostic value is best realized when interpreted in the context of additional clinical, biochemical, and imaging data. (28) From a therapeutic perspective, HRV analysis offers a non-invasive means of monitoring treatment effects and evaluating patient response to interventions targeting autonomic regulation. (28,29) These interventions include pharmacological therapies (such as β-blockers, ivabradine), exercise-based cardiac rehabilitation, vagus nerve stimulation, biofeedback techniques, and lifestyle modifications such as mindfulness training and stress reduction. (29) Improvements in HRV have been consistently associated with better clinical outcomes and reduced mortality, particularly in patients with HF undergoing different rehabilitation programs. Furthermore, the integration of HRV into existing risk stratification frameworks, together with established biomarkers such as troponin and modern imaging techniques, may enhance early recognition of high-risk patients and support personalized therapeutic decision-making. (30)
HEART RATE RECOVERY
Heart rate recovery (HRR) is a clinically relevant, non-invasive marker of ANS function, particularly with regard to the dynamic reactivation of parasympathetic (vagal) tone following exercise cessation. It is defined as the rate at which HR declines from peak exertion to a specific time point during the recovery phase. Physiologically, HRR is thought to reflect the interplay between immediate vagal reactivation and gradual sympathetic withdrawal, primarily mediated via baroreflex mechanisms and modulation of SNA. (31) HRR appears to consist of two distinct phases: initial rapid decline in HR within the first minute after exertion, predominantly driven by parasympathetic reactivation, followed by a slower, progressive decline, influenced by continued parasympathetic activity and progressive sympathetic withdrawal. This biphasic pattern enables HRR to capture both acute and sustained aspects of autonomic recovery. (31,32) The most commonly used and clinically validated time point is HRR measured at 1 minute after exertion, where a decrease of ≤12 beats per minute is generally considered abnormal. This threshold has been associated with an increased risk of adverse CV outcomes and is widely accepted in clinical and research settings. (33,34) However, normative values may vary depending on age, sex, and CV fitness level, as well as pharmacological treatment and comorbid conditions. Notably, trained individuals typically exhibit HRR values exceeding 20-30 beats per minute at 1 minute after exertion. Therefore, HRR interpretation should be contextualized using population- and condition-specific reference ranges whenever possible. (35) HRR may only be reliably assessed after standardized exercise testing, typically conducted using a treadmill or cycle ergometer with a clearly defined point of exercise termination. Such exercise testing also allows for the evaluation of HR response to exertion, which is an important indicator of CV function and autonomic regulation (Figure 2), and commonly includes BP monitoring. Extended measurements of HR at 2, 3, or 5 minutes after exertion may provide further insight into autonomic dissonance in case of a delay in parasympathetic reactivation. (35,36)
From a prognostic standpoint, HRR has demonstrated moderate sensitivity and specificity in predicting all-cause mortality, SCD, and incident coronary artery disease (CAD), particularly when used in combination with other clinical indicators. (35-37) Impaired HRR is believed to reflect sustained sympathetic activation and/or blunted parasympathetic reactivation, both of which are known contributors to adverse CV events. The lack of adequate vagal tone after exertion removes a key antiarrhythmic buffer, increasing the risk of ventricular arrhythmias, impaired coronary perfusion, and SCD, all of which contribute to poor prognosis. (38,39) For example, among patients with documented CAD, a robust meta-analysis encompassing more than 2,400 participants demonstrated that attenuated HRR at 1 minute after exertion (thresholds ranging from ≤12 to ≤21 beats per minute) was strongly predictive of all-cause mortality, with a pooled hazard ratio of around 5.8. These findings highlight the prognostic importance of even subtle impairments in autonomic recovery after exertion. (40) More recent large-scale prospective studies have further reinforced and refined the prognostic relevance of HRR across diverse populations. In particular, a 2024-2025 prospective cohort study including nearly 10,000 adults undergoing standardized cycle ergometer testing demonstrated that a blunted 1-minute HRR, defined as a value below the 5th percentile, was independently associated with an increased risk of all-cause mortality (hazard ratio approximately 1.70). Notably, this association remained robust even in individuals with preserved exercise capacity, highlighting HRR as a sensitive marker of early autonomic impairment in populations traditionally considered to be at lower CV risk. (41)
It should be emphasized that HRR is not a standalone diagnostic tool but rather a prognostic biomarker that gains clinical value when interpreted within a comprehensive assessment of autonomic function. Reduced HRR has been consistently associated with adverse clinical outcomes, including HTN, progression of CAD, and higher incidence of CV events in HF, as demonstrated across multiple observational studies and clinical reviews. (36,42,43) These individuals typically exhibit delayed autonomic recovery after exertion, characterized by attenuated parasympathetic reactivation and sustained sympathetic activity, in contrast to healthy individuals and endurance-trained athletes, who demonstrate rapid and efficient vagal reactivation (Figure 3). Given its prognostic relevance and simplicity of assessment, several authors advocate for the routine incorporation of HRR assessment into clinical exercise testing protocols. (42,44)
From a therapeutic perspective, HRR serves as a sensitive indicator of autonomic recovery and cardiorespiratory fitness, useful for monitoring treatment efficacy in CV disease. Evidence from larger observational cohorts and more methodologically robust studies consistently links improvements in HRR with more favorable clinical outcomes, including reduced hospitalization rates and lower mortality. In contrast, some earlier interventional studies reporting improvements in HRR following cardiac rehabilitation were conducted in relatively small, single-center cohorts with consecutive patient enrollment, which warrants cautious interpretation due to the limited external validity of these findings. (45) Nonetheless, exercise-based cardiac rehabilitation has been shown to improve HRR across different study designs, with combined exercise training and β-blocker therapy yielding greater benefits than pharmacological treatment alone. (46) While these studies provided important proof-of-concept data, their limited sample size and study design may constrain the generalizability of the findings.
Additional studies further supports the responsiveness of HRR to therapeutic modulation. Controlled physiological studies examining the effects of pharmacological heart rate-lowering agents, such as β-blockers and ivabradine, as well as vagal nerve stimulation, have demonstrated favorable shifts in autonomic recovery indices, including HRR. (47)
Finally, evidence from observational and rehabilitative cohorts, including earlier but methodologically sound longitudinal studies, suggests that structured exercise training is consistently associated with improvements in HRR over time. (48) Although some of these studies predate contemporary rehabilitation protocols, their findings remain relevant and support the role of HRR as a dynamic marker of therapeutic response.
AMBULATORY AND HOME BLOOD PRESSURE MONITORING
Precise evaluation of BP is crucial for both the diagnosis and effective management of HTN. (49) Although in-office BP measurements are still widely used in routine clinical settings, their reliability can be compromised by factors such as the white coat effect or masked HTN. (50) To overcome these limitations, BP monitoring outside the clinical environment, such as home blood pressure monitoring (HBPM) and 24-hour ambulatory blood pressure monitoring (ABPM), may provide more consistent and clinically meaningful information. ABPM, which continuously records BP throughout a 24-hour period during a patient’s typical daily routine and sleep, offers valuable data on circadian BP fluctuations and the functional status of the ANS. (51,52) ABPM is performed using a portable device worn on the patient’s arm, which automatically inflates at preset intervals, and can be conducted either in a clinical setting or at home during normal daily activities. (52) This method allows for the measurement of BP in relation to the day-night rhythm and its variation, providing a more complete picture of BP elevation and the specific times of day when it occurs (Figure 4). In the context of autonomic dysfunction, HR measurements combined with BP values can provide further insights into autonomic function, especially when supplemented by additional analysis of HRV parameters, as previously discussed. It is also important to consider BP patterns during different times of the day. Specific patterns, such as normal nocturnal dipping, absence of dipping (non-dipping), reverse dipping (riser pattern), and abnormal morning BP surges are recognized indicators of ANS function. These patterns reflect the balance and interaction between sympathetic and parasympathetic activity. (53)
Clinically, individuals who lack the normal nocturnal fall in BP (non-dippers), often exhibit heightened nocturnal sympathetic activity accompanied by diminished vagal tone. This association has been supported primarily by data from large, well-characterized cohort studies, such as the African Prospective study on the Early Detection and Identification of Cardiovascular disease and Hypertension (African-PREDICT), which demonstrated that non-dipping BP patterns and blunted morning BP surges are associated with reduced BRS and lower HRV, both markers of ANS dysfunction. (54) Further insight into the relationship between circadian BP variation and autonomic regulation has been provided by population-based observational studies using ABPM. For example, the Hypertension and Ambulatory Blood Pressure Monitoring Project in Japan (HI-JAMP) study showed that BP responsiveness to daily physical activity (also known as actisensitivity) differs across dipping phenotypes, including non-dipping, extreme dipping, and reverse dipping patterns, reflecting heterogeneity in autonomic modulation. (55) In addition to its established role in the diagnosis of HTN, 24-hour ABPM may provide clinically useful, non-invasive insights into autonomic function, especially when integrated with other autonomic indices. This is particularly relevant in conditions such as diabetes, obstructive sleep apnea, and HF, where ANS imbalance frequently contributes to disease progression and CV risk. (56) Therefore, it can be concluded that ABPM should be more broadly implemented in clinical practice, not only as a diagnostic standard for HTN, but also as a supplementary tool for evaluating ANS function in various cardiometabolic and neuroregulatory disorders. (57) This may also have important prognostic implications, as autonomic imbalance contributes to end-organ damage, arrhythmogenesis, and poor CV outcomes. (52,58)
In terms of HTN treatment, HBPM is valuable for long-term follow-up and promoting patient adherence. However, it does not capture nocturnal BP or circadian variability, thereby limiting its utility in assessing ANS function. (59) In contrast, ABPM plays a central role in evaluating the effectiveness of antihypertensive therapy by providing continuous 24-hour data, including nighttime BP profiles and short-term variability. This allows clinicians to identify inadequate nocturnal dipping, optimize medication timing and combinations, and better individualize antihypertensive regimens. (60) ABPM is particularly beneficial in managing conditions associated with increased CV risk and requiring strict BP control, such as chronic kidney disease, (61) left ventricular hypertrophy, (62) and HF. (63) When used alongside tools such as ECG, HRV analysis, or exercise stress testing, ABPM enhances the overall assessment of ANS function. (64) In summary, ABPM provides substantial clinical value in both HTN management and the evaluation of ANS function, supporting a more individualized and pathophysiology-based approach to care. In addition to resting and ambulatory measurements, exaggerated blood pressure response to exercise (EBPR) provides a dynamic assessment of vascular and autonomic reactivity under physiological stress. EBPR, characterized by an abnormally steep rise in systolic BP during graded exercise, reflects excessive sympathetic activation, impaired baroreflex buffering, and increased arterial stiffness. (65) Although it is not part of standard BP monitoring, integrating EBPR findings with ABPM or HRV data offers complementary insights into autonomic-hemodynamic coupling and early CV risk. (66) Individuals exhibiting EBPR often demonstrate altered nocturnal BP profiles or non-dipping patterns on ABPM, underscoring the shared autonomic pathways underlying both exercise and circadian BP regulation. (66,67)
BAROREFLEX SENSITIVITY
Baroreflex sensitivity denotes the ability of the ANS to modulate HR in response to acute changes in BP, thereby contributing to short-term CV stability. It reflects the functional integrity of the baroreflex arc, particularly the sensitivity of baroreceptors located in the carotid sinus and aortic arch, which detect fluctuations in arterial pressure and trigger appropriate autonomic adjustments. (68) Assessment of BRS can be performed through pharmacological or non-pharmacological approaches. The pharmacological method typically involves intravenous administration of vasoactive substances such as phenylephrine to provoke transient changes in BP, with concurrent measurement of the resulting reflexive HR responses. (69) Non-pharmacological assessments include standardized maneuvers and analytical techniques, such as the Valsalva maneuver, application of neck chamber suction or pressure to stimulate baroreceptors, and spontaneous sequence analysis. (69,70) The latter method identifies naturally occurring sequences of systolic BP and RR interval changes on a beat-to-beat basis, allowing for non-invasive and continuous estimation of baroreflex gain under resting conditions. (71) Data are typically analyzed using linear regression (slope of RR interval vs. systolic BP) or spectral analysis in the frequency domain, quantifying the coherence between BP and HR oscillations (Figure 5). Newer methods include transfer function analysis and model-based approaches for improved resolution. (72)
Recent research highlights the growing clinical relevance of BRS as a non-invasive marker of ANS function and a powerful prognostic indicator in CV disease. (72,73) Giannoni et al. (2022) found that BRS, alongside chemoreflex sensitivity, independently predicted adverse outcomes in patients with chronic HF. Patients with impaired BRS exhibited significantly higher rates of CV mortality and hospitalization, supporting its potential utility in risk stratification and clinical management. While this study was methodologically robust, it is important to note that the findings were derived from specialized HF populations and may require cautious generalization to broader patient groups. (74) Zhou et al. (2023) used population-based data from the Maastricht Study and demonstrated an inverse relationship between BRS and both systolic BP and BP variability. These findings suggest that reduced BRS contributes to impaired CV regulation and may be involved in the early pathophysiological processes leading to HTN. (75) These findings emphasize that BRS serves not only as a marker of autonomic function but also as a potential target for preventive strategies.
BRS has also been associated with orthostatic hypotension (OH), a condition characterized by an excessive drop in BP upon standing, often due to inadequate autonomic compensation. In a recent observational study, Klop et al. showed that BRS declines with age and is markedly reduced in individuals exhibiting OH. This reduction was linked to impaired CV adaptation and attenuated cerebral oxygenation during postural transitions. While the study provides valuable mechanistic insight, its cross-sectional design warrants cautious interpretation regarding causality. Nevertheless, these data underscore the potential clinical value of BRS assessment in identifying patients at elevated risk of OH-related complications. (76)
Building on the prognostic relevance of BRS in chronic HF, recent therapeutic research has explored modulation of autonomic tone as a potential intervention. (74,77) Gentile et al. (2024) reviewed the emerging role of vagus nerve stimulation in patients with HF, highlighting that impaired BRS reflects reduced parasympathetic (vagal) activity and excessive sympathetic drive, both key contributors to disease progression. By enhancing vagal efferent activity, vagus nerve stimulation may improve BRS, reduce arrhythmic risk, and positively influence cardiac function and remodeling. These studies support the hypothesis that BRS is not only a biomarker of autonomic dysfunction but also a potentially modifiable parameter with therapeutic implications. (77)
Overall, BRS is a clinically valuable, non-invasive marker of autonomic regulation, offering high specificity and good sensitivity for detecting autonomic dysfunction, particularly in populations with HF. When combined with complementary autonomic tests such as HRV and deep breathing, BRS contributes to a comprehensive evaluation of autonomic function. (78,79)
TILT-TABLE TESTING
Tilt-table testing (TTT) is a standardized, non-invasive diagnostic procedure used to evaluate the ANS, particularly in the context of unexplained syncope, orthostatic intolerance, and autonomic dysfunction. (80) It assesses the CV response to controlled postural changes, typically from a supine to an upright position, thereby simulating orthostatic stress under monitored conditions. (80,81) In clinical practice, the procedure involves securing the patient to a motorized table that passively tilts them from a horizontal (supine) position to an angle between 15° and 75°, typically maintained for 20 to 45 minutes. Continuous ECG and non-invasive BP monitoring are used to record HR and BP responses throughout the test (Figure 6). (81,82) In some protocols, pharmacological provocation with agents such as nitroglycerin may be employed to increase diagnostic yield, especially when initial passive tilt yields inconclusive results. (82)
The primary diagnostic value of TTT lies in its ability to reproduce symptoms and detect abnormal autonomic responses, particularly in the evaluation of neurally mediated syncope (vasovagal syncope), postural orthostatic tachycardia syndrome, and orthostatic intolerance. (81-83) It helps distinguish between reflex-mediated syncope from other causes of transient loss of consciousness, such as arrhythmias, CAD, or structural heart disease, which may not be evident in routine testing. (84,85) TTT was formally introduced into clinical practice in the late 1980s and has since become a well-established diagnostic modality in the evaluation of syncope and autonomic dysfunction. (86) Its diagnostic sensitivity ranges from 60% to 85%, while specificity varies between 90% and 95%, depending on the patient population and the protocol employed. Although a negative tilt-table test does not definitively exclude reflex syncope, a positive test provides strong diagnostic support, particularly when symptom reproduction is accompanied by characteristic hemodynamic changes. (86-88) While false positives may occur in healthy individuals, patients with a history of reflex syncope typically present a shorter time to syncope and a more pronounced, sustained drop in BP. False negatives have been reported in up to 30% of cases. In such cases, prolonged ECG monitoring may subsequently uncover a cardioinhibitory mechanism. (89)
Tilt-table testing enables the assessment of OH under controlled conditions by applying sustained orthostatic stress while continuously monitoring beat-to-beat BP and HR. (90) It can reveal a pathological decrease in systolic and/or diastolic BP that meets diagnostic criteria (≥20/10 mmHg), even in patients with inconclusive bedside orthostatic measurements. This makes it especially useful for identifying neurogenic OH, distinguishing it from other causes of orthostatic intolerance, and for detecting delayed forms that manifest only after prolonged upright posture. (91) Tilt testing is particularly indicated in cases where symptoms such as dizziness, presyncope, or unexplained falls suggest an orthostatic mechanism, but conventional standing tests are non-diagnostic, as well as in patients unable to perform active stand testing due to frailty or mobility limitations. (92) In such contexts, TTT is recommended in conjunction with other autonomic function tests, including BRS assessment, HRV analysis, the Valsalva maneuver, and deep breathing tests in order to form a comprehensive autonomic profile. When interpreted in conjunction with clinical history and other test results, it offers valuable insights on the autonomic regulation of CV function and supports accurate diagnosis, risk stratification, and management decisions. (92-94)
Alternative and emerging methods for assessing autonomic nervous system function
Although many of these methods were originally developed or primarily applied in neurology, endocrinology, or research settings, their ability to quantify autonomic regulation of CV function makes them particularly relevant in cardiology. These additional methods can be combined with core autonomic function tests, such as HRV, BRS, or tilt-table testing to provide complementary insights into HR regulation, BP variability, and autonomic balance (Table 2). Such parameters are significantly involved in the pathophysiology and risk assessment of various cardiac conditions, including syncope, arrhythmias, HF, and post-MI outcomes. (95,96)
DEEP BREATHING TEST
The deep breathing test is a simple, noninvasive clinical method for evaluating cardiovagal (parasympathetic) function. During the procedure, the patient performs deep, paced breathing at a fixed rate (typically 5 to 6 breaths per minute), while continuous ECG monitoring captures the heart’s beat-to-beat variability. This allows for assessment of respiratory sinus arrhythmia, the physiological fluctuation in HR between inspiration and expiration. The key diagnostic metric is the difference between the maximal and minimal HR within each respiratory cycle (or the expiratory/inspiratory ratio, E/I ratio), computed over several respiratory cycles and often averaged across six consecutive breaths. (97) Some scientists emphasize that HRV during deep breathing is a highly sensitive marker of vagal modulation, providing early detection of parasympathetic dysfunction even before overt clinical signs of autonomic impairment become apparent. (98) Han and Zhang (2025) proposed a novel heartbeat-respiration coupling metric that offers superior sensitivity for detecting shifts in autonomic balance beyond conventional HRV measures. Their findings support the clinical utility of this approach as a non-invasive tool for evaluating autonomic function in clinical cardiology. (99)
VALSALVA MANEUVER
The Valsalva maneuver is a well-established, non-invasive method for assessing autonomic function, particularly the integrity of baroreflex control and cardiovagal tone. (100) By inducing forced expiration against a closed airway, this maneuver provokes characteristic changes in HR and BP across four physiological phases. (100,101) The Valsalva ratio, defined as the ratio of peak HR during strain to the lowest HR during recovery, is a key index of parasympathetic function, whereas accompanying BP responses provide insight into sympathetic vasomotor control. (102) Its clinical relevance in cardiology was demonstrated by Felker et al. (2006), who introduced it as a novel, non-invasive method for assessing cardiac filling pressures in HF. (103) Although its cardiological application has since received limited attention in research, Park et al. (2023) more recently underscored its value in identifying late-onset delayed orthostatic intolerance, a subtle yet clinically significant manifestation of autonomic dysfunction. (104) The Valsalva maneuver can be routinely performed alongside other autonomic function tests, such as HRV, BRS, deep breathing, and tilt-table testing, in order to enable a comprehensive assessment of ANS regulation in cardiology. (102,105)
ACTIVE STAND TEST (ORTHOSTATIC BLOOD PRESSURE MEASUREMENT)
The orthostatic (active standing) test provides a simple, non-invasive means of evaluating autonomic CV regulation and the capacity of the ANS to preserve hemodynamic stability during postural transition. After at least 5 minutes of supine rest, the patient promptly stands, and continuous beat-to-beat measurements of HR and BP are recorded over 3 to 10 minutes, according to the respective protocol or guideline. (106) However, continuous beat-to-beat monitoring is rarely performed in routine clinical practice. Instead, a standardized bedside protocol using intermittent oscillometric BP measurements performed at defined intervals after standing is commonly applied, as recommended by current guidelines. Although this approach offers lower temporal resolution and sensitivity compared to beat-to-beat methods, it remains more feasible and widely applicable in everyday clinical settings. (107) This test is primarily used to identify autonomic dysfunctions such as OH. (107,108) In cardiology, it is particularly relevant for uncovering baroreflex impairment or sympathetic failure contributing to syncope, dizziness, falls, or other orthostatic symptoms. Conversely, orthostatic hypertension, defined as an excessive increase in systolic BP upon standing, has also gained attention as a potential marker of sympathetic overactivity and increased CV risk. (109) Beyond the diagnostic classification, Gronwald et al. (2024) have highlighted the additional value of active stand tests in assessing HRV under orthostatic stress, providing insight into parasympathetic reactivity and recovery dynamics. (110) Despite its practicality, the orthostatic test has notable limitations in detecting certain forms of autonomic dysfunction. Kirbiš et al. (2013) pointed out that the active stand test may miss delayed or subtle autonomic responses, which are more reliably detected with tilt-table testing or 24-hour ABPM. Limitations also include reduced reproducibility due to individual variability, muscle activity, and respiratory influence, as well as a practical constraint in patients with frailty or impaired mobility. (111) Although it lacks the standardization and controlled gravitational stimulus of passive tilt testing, the orthostatic test remains a valuable component of comprehensive autonomic evaluation, particularly when interpreted in conjunction with complementary measures such as HRV, BRS, Valsalva maneuver, and TTT findings. (107,108,112)
HEART RATE ACCELERATION AND DECELERATION CAPACITY
Advanced HRV analysis using phase-rectified signal averaging yields acceleration capacity (AC) and deceleration capacity (DC), which reflect sympathetic and parasympathetic HR modulation, respectively. Although primarily used in research, these metrics are emerging as sensitive markers of autonomic imbalance. DC, in particular, has shown prognostic value in patients with HF, predicting both arrhythmic and all-cause mortality. (113) More recently, AC and DC have also been associated with circadian patterns of BP in essential HTN, highlighting their potential relevance in broader CV regulation. (114)
MICRONEUROGRAPHY
Microneurography is an invasive technique that enables direct recording of sympathetic nerve activity in humans by inserting a microelectrode into a peripheral nerve, typically the peroneal nerve. It provides real-time measurements of muscle sympathetic nerve activity, providing unique insights into ANS function. (115) In cardiology, microneurography has been used to investigate sympathetic overactivity in conditions such as HF, HTN, and arrhythmias. It allows precise assessment of sympathetic outflow, complementing indirect measures such as HRV. Although technically demanding and limited to specialized research centers, its potential in detecting autonomic dysfunction in CSV disease could be significant. Future research combined with noninvasive markers of ANS activity could enhance our understanding of autonomic contributions to CV risk. (116)
SYMPATHETIC SKIN RESPONSE
The sympathetic skin response (SSR) measures changes in electrical potential on the surface of the skin triggered by sweat gland activation, reflecting sudomotor sympathetic activity. As a noninvasive and easily applicable test, SSR is primarily used to assess small-fiber autonomic neuropathies. (117) In cardiology, SSR may help identify subtle autonomic dysfunction not detected by standard CV reflex tests. Studies have demonstrated abnormal SSR findings in patients with peripheral arterial occlusive disease, indicating altered sympathetic function. Similar changes have been reported in cardiac conditions such as HF and syncope. (118) Although still mainly a research tool, SSR could support autonomic profiling in CV disease when used alongside other assessments.
SKIN CONDUCTANCE RESPONSE
Skin conductance response (SCR) is a noninvasive measure of sudomotor sympathetic activity, reflecting small-fiber autonomic function. In cardiology, it may aid in detecting autonomic dysfunction in conditions such as HF, syncope, or diabetic neuropathy. (119) The efficacy and reproducibility of SCR make it a useful complementary diagnostic tool, though further validation in CV populations is needed.
Clinical utility and implementation of autonomic nervous system testing in everyday practice
The inclusion of ANS evaluation in CV assessment offers considerable diagnostic and prognostic value. Non-invasive modalities such as HRV, HRR, BRS, TTT, and ABPM each provide complementary insights into autonomic regulation and CV risk (Table 3). The first formal recommendations for HRV analysis were issued in the mid-1990s by the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology, establishing standardized metrics for time- and frequency-domain measures. Since then, methodological advancements, digital analytics, and expanding clinical evidence have refined HRV interpretation and broadened its relevance across CV and systemic diseases. (120) Despite this progress, HRV and other ANS-related markers have yet to be fully integrated into contemporary European or American clinical guidelines.
The 2021 consensus statement by Cheshire et al., endorsed by the American Autonomic Society and other expert bodies, underscored the pressing need for standardization and consensus in ANS testing. It recommended that autonomic evaluation incorporate a combination of validated measures addressing cardiovagal, adrenergic, and sudomotor domains, interpreted within the broader clinical context rather than as isolated diagnostics. The document further highlighted the importance of methodological harmonization, establishment of normative reference data, and expert oversight to ensure accuracy and comparability across institutions. Establishing such consensus-based frameworks represents a crucial step toward integrating ANS testing into routine cardiological practice. (121) In line with these recommendations, Verdugo and Matamala (2021) observed that, despite the long-standing clinical use of autonomic tests, their widespread implementation has been limited by the lack of standardized procedures and interpretative criteria. They emphasized that the introduction of internationally accepted standards now provides a coherent framework for performing and interpreting autonomic testing, thereby improving diagnostic reliability, cross-center comparability, and clinical applicability. The authors advocate for the systematic integration of standardized autonomic assessments into CV evaluation to enhance diagnostic precision, therapeutic guidance, and research consistency. (122)
The 2024 update on Clinical Neurocardiology by Ajijola et al. (2025) highlights that ANS dysfunction represents a fundamental pathophysiological basis of many CV diseases, including arrhythmias, HF, and post-infarction syndromes. The authors emphasize that advances in neuroanatomy, neurophysiology, and neuromodulation are increasingly shaping CV therapeutics, gradually shifting focus from symptomatic management towards targeted modulation of neural control. Autonomic testing and neuromodulatory strategies are viewed as promising tools for refined risk stratification and individualized therapy, although their clinical translation is still evolving. The paper calls for the development of standardized biomarkers, improved sensor technologies, and closed-loop systems linking ANS assessment with therapy. Finally, it stresses the importance of interdisciplinary collaboration to establish unified frameworks for integrating autonomic assessment and neuromodulatory treatment into mainstream clinical practice. (123) Ensuring precision and accuracy remains fundamental in autonomic testing, as emphasized by the Policy Department of the American Association of Neuromuscular and Electrodiagnostic Medicine, which established core principles for standardized performance, patient safety, and reliable interpretation. (124) With the rapid expansion of digital health technologies and wearable monitoring systems, autonomic measures, particularly HRV, can now be seamlessly integrated into clinical workflows, enhancing early detection of autonomic dysfunction and enabling longitudinal monitoring of therapeutic efficacy. (125)
Limitations
Several limitations of this article should be acknowledged. First, as a narrative synthesis rather than a systematic review or meta-analysis, the selection of studies may be subject to bias, potentially limiting reproducibility. Second, the reviewed literature exhibits substantial heterogeneity in study design, methodological rigor, patient populations, ANS testing protocols, data acquisition methods, analytical approaches, and clinical endpoints. This variability spans small observational studies, single-center interventional trials, large prospective cohorts, and mechanistic or experimental studies, which complicates direct comparisons and constrains the ability to draw definitive conclusions regarding clinical thresholds or standardized applications. Third, while many studies provide valuable physiological and prognostic insights into autonomic markers such as HRV, HRR, ABPM-derived measures, and BRS, the overall strength of the evidence is heterogeneous. Larger studies and meta-analyses generally support the clinical utility of these markers, whereas findings from smaller, single-center, or early-phase interventional studies should be interpreted with caution due to limited generalizability. Additionally, several studies focus on surrogate endpoints rather than hard CV outcomes, which may affect the translational relevance of some findings. Finally, rapid technological advances and evolving analytical techniques in ANS assessment may limit the generalizability of older studies over time. Taken together, although the convergence of findings across diverse populations supports the clinical relevance of autonomic markers for risk stratification, prognostic assessment, and monitoring of therapeutic interventions, readers should consider study design, methodological quality, and population characteristics when interpreting results and applying them to clinical practice. Future research should aim towards standardized protocols, larger multicenter studies, and clinically meaningful endpoints to strengthen the translational value of ANS testing.
Conclusion
Early recognition of autonomic dysfunction enables targeted interventions, such as optimization of pharmacotherapy, lifestyle modification, or cardiac rehabilitation, which may reduce arrhythmic risk, improve BP control, and enhance clinical outcomes. Incorporating ANS measures into routine cardiology practice can support a more comprehensive and integrative approach to CV care, bridging functional and structural assessment. Autonomic function testing offers unique physiological insights that cannot be captured by conventional hemodynamic or imaging methods, providing a functional dimension that complements structural diagnostics and aids in personalized, patient-centered management. The evidence supporting these applications spans a wide range of study designs, from small observational cohorts to large prospective studies and consensus-based recommendations. While many studies provide robust insights on HRV, HRR, BRS, and other autonomic markers, the overall strength of evidence remains heterogeneous. Variations in methodological quality, sample size, study design, patient populations, and outcome measures limit consistency across studies and should be considered when interpreting clinical implications. Meta-analyses and larger cohort studies generally provide the most reliable data, whereas findings from smaller or single-center studies warrant cautious interpretation. Despite these limitations, the convergent direction of evidence across diverse populations supports the clinical relevance of autonomic markers for risk stratification, prognostic assessment, and monitoring of therapeutic interventions. Broader implementation in clinical practice will require standardized protocols, professional training, and internationally harmonized guidelines to optimize test performance, result interpretation, and integration into CV care. Future research should prioritize larger, multicenter studies with clinically meaningful endpoints to strengthen the translational value of ANS testing and further establish its role in modern cardiology.