Autonomic dysfunction and insulin resistance in type 2 diabetes mellitus with hypoglycemia unawareness: an observational study

Abstract

Purpose

To evaluate the association between hypoglycemia unawareness, cardiac autonomic function, and insulin resistance in individuals with type2 diabetes, and explore potential correlations between autonomic dysfunction and metabolic parameters.

Methods

50 individuals with type2 diabetes (age 50–65 years, duration >5 years) were recruited and classified into two groups using Clarke’s Hypoglycemia Awareness Questionnaire: hypoglycemia unawareness group (n = 25) and controls (n = 25). Standardized cardiovascular autonomic function tests (AFTs) and short-term heart rate variability (HRV) analyses were performed. Autonomic dysfunction was graded using Ewing’s, Bellavere’s, and All India Institute of Medical Sciences AFT Lab criteria.

Results

The hypoglycemia unawareness group exhibited significantly lower Valsalva ratio (1.49 ± 0.31) vs. the control group (1.80 ± 0.42) (P = 0.004), and reduced diastolic blood pressure (BP) response during the cold pressor test (10.92 ± 9.81 mmHg) vs. the control group (18.36 ± 8.50 mmHg) (P = 0.001). A greater fall in systolic BP during postural change was also noted (11.08 ± 6.38 mmHg) vs. the control group (7.64 ± 3.53 mmHg) (P = 0.037). HRV indices like SD of all N–N intervals and total power were reduced in hypoglycemia-unawareness individuals, indicating impaired sympatho-vagal modulation. Fasting glucose was significantly higher in hypoglycemia-unawareness individuals (114.36 ± 11.81) vs. the control group (106.44 ± 13.11) (P = 0.021), but no significant difference in homeostatic model assessment for insulin resistance was observed. Serum insulin levels showed negative correlations with sympathetic function measures.

Conclusion

Hypoglycemia unawareness in T2DM is associated with combined sympathetic and parasympathetic dysfunction and reduced HRV, consistent with a hypoglycemia-associated autonomic failure cycle. Routine autonomic assessment may aid in early detection, risk stratification, and prevention of adverse cardiovascular outcomes in this high-risk subgroup.

Introduction

Diabetes mellitus is a growing global health challenge, with the International Diabetes Federation estimating that over 537 million adults worldwide are currently living with diabetes, a figure expected to rise to 643 million by 2030 [1]. India alone accounts for over 74 million cases, ranking second globally in diabetes prevalence [2]. Type 2 diabetes mellitus (T2DM) constitutes more than 90% of these cases and is associated with a broad spectrum of acute and chronic complications. Among its less visible yet clinically significant complications is diabetic autonomic neuropathy, particularly cardiac autonomic neuropathy (CAN), which increases the risk of cardiovascular morbidity and mortality, often without overt symptoms [3]. Despite its serious implications, CAN remains underdiagnosed because of its subclinical progression and the lack of routine testing.

CAN is characterized by early alterations in autonomic balance, such as reduced parasympathetic activity and enhanced sympathetic dominance [4]. These changes predispose individuals to cardiovascular instability, silent ischemia, and arrhythmias [5,6]. Importantly, autonomic dysfunction may also contribute to another high-risk and underrecognized complication in T2DM hypoglycemia unawareness. Hypoglycemia unawareness is defined as the inability to recognize the early adrenergic symptoms of hypoglycemia, which delays corrective action and increases the risk of severe hypoglycemia, seizures, loss of consciousness, and even death [7]. Approximately 25–30% of individuals with long-standing T2DM, especially those on insulin therapy, experience hypoglycemia unawareness, complicating glycemic control, and diminishing quality of life, and hypoglycemia unawareness and autonomic dysfunction are also thought to be patho-physiologically interconnected [8]. On one hand, recurrent hypoglycemic episodes blunt the normal sympathetic and hormonal counter-regulatory responses a condition termed hypoglycemia-associated autonomic failure (HAAF) as shown in Fig. 1. On the other hand, individuals with pre-existing autonomic neuropathy may already exhibit diminished sympathoadrenal responses, increasing their susceptibility to hypoglycemia unawareness. This bidirectional relationship suggests a potential vicious cycle between autonomic failure and impaired hypoglycemia awareness [9].

Fig. 1.

Interplay between hypoglycemia unawareness and cardiac autonomic neuropathy in T2DM. CPT, cold pressor test; HRV, heart rate variability; SDNN, SD of all N–N intervals; T2DM, type 2 diabetes mellitus.

Another important metabolic disturbance in T2DM is insulin resistance. Insulin resistance is not only central to the pathogenesis of T2DM [10] but is also associated with systemic inflammation, endothelial dysfunction, and altered autonomic tone [11]. Some studies have reported an inverse association between insulin resistance and heart rate variability (HRV), a noninvasive marker of autonomic function [12–14]. However, evidence remains inconclusive, and the potential interplay between insulin resistance, hypoglycemia unawareness, and autonomic dysfunction is not well understood. While individual studies have explored hypoglycemia unawareness, insulin resistance, and autonomic dysfunction in isolation, few have examined all three simultaneously using standardized autonomic function tests (AFTs), validated clinical questionnaires, and objective metrics such as HRV. A deeper understanding of these interrelationships may facilitate risk stratification and enable earlier identification of T2DM individuals vulnerable to severe hypoglycemia or cardiac complications.

Therefore, the present study was undertaken to investigate the association between hypoglycemia unawareness, autonomic dysfunction, and insulin resistance in individuals with T2DM. The primary aim was to compare AFT results and levels of insulin resistance between individuals with and without hypoglycemia unawareness. Specifically, the objectives were: (a) to assess and compare autonomic function using standardized AFTs; (b) to evaluate insulin resistance using the homeostatic model assessment for insulin resistance (HOMA-IR); and (c) to explore potential correlations between hypoglycemia unawareness, CAN, and insulin resistance in this patient population.

Methods

Study design and setting

This was an observational study conducted over 18 months (November 2019 to May 2021) in the Autonomic Function Laboratory, Department of Physiology, in collaboration with the Departments of Medicine and Laboratory Medicine, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi.

Study population

A total of 50 individuals with T2DM, aged between 50 and 65 years, and with a disease duration of more than 5 years, were recruited from the Medicine Outpatient Department. Based on their history and assessment using Clarke’s Hypoglycemia Awareness Questionnaire [15], the participants were divided into two groups:

• 1.Hypoglycemia unawareness group: 25 individuals with a history of one or more episodes of hypoglycemia unawareness.
• 2.Control group: 25 individuals without any prior episodes of hypoglycemia unawareness.

Participants in both groups were matched for age, sex, and duration/type of diabetes treatment (oral hypoglycemic agents or insulin therapy).

Sample size calculation

The sample size was calculated using the formula:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{N}\mathrm{A}=\mathrm{K}\mathrm{N}\mathrm{B}}\\ =[\{\mathrm{P}\mathrm{A}\left(1\mathrm{P}\mathrm{A}\right)+\mathrm{P}\mathrm{B}\left(1\mathrm{P}\mathrm{B}\right)\}/K\\ \times {(\{Z\left(\alpha /2\right)+Z\left(1\beta \right)\}/\left\{\mathrm{P}\mathrm{A}\mathrm{P}\mathrm{B}\right\})}^2],\end{array}}$$

based on the proportions of individuals with hypoglycemia unawareness and autonomic dysfunction as described by the study of Ryder et al. [16] with a 95% confidence interval, 80% power, ±20% margin of error, and 10% allowance for nonresponse, the minimum required sample size was 19 per group. Considering attrition, uninterpretable data, and drop-out rate, the sample size for our study was taken as 25 subjects in each group.

Ethics statement

All participants were provided with a patient information sheet, and written informed consent was obtained. Ethical clearance was granted by the Institutional Ethics Committee of VMMC & Safdarjung Hospital with number (IEC/VMMC/SJH/2019-10/257). The procedures adhered to the guidelines outlined in the Declaration of Helsinki 2013.

Inclusion and exclusion criteria

Inclusion criteria

• 1.Diagnosed cases of T2DM aged 50–65 years.
• 2.Disease duration is more than 5 years.
• 3.With or without hypoglycemia unawareness, as per Clarke’s questionnaire.

Exclusion criteria

• 1.Documented cardiovascular, any other endocrine, or autoimmune disorders.
• 2.History of arrhythmia, valvular disease, or congenital heart disease (as confirmed by ECG/echocardiography).
• 3.Current febrile illness, neuropsychiatric disorder, or respiratory disease.
• 4.Chronic smokers, substance abusers, or patients on hormone replacement therapy.

Study protocol

All assessments were conducted between 9 : 00 and 11 : 30 a.m. in a temperature-controlled lab (23–25 °C). Participants were asked to: Avoid caffeine or tea on the day of testing, have a light breakfast at least 2 h prior, and rest quietly for 20 min before testing. Clinical history and anthropometric measurements (height, weight, and BMI) were recorded. A fasting blood sample (after 8 h of fasting) was collected for measurement of plasma glucose and insulin [17].

Autonomic function testing

Six standard cardiovascular autonomic reflex tests were performed based on the All India Institute of Medical Sciences (AIIMS) AFT Lab protocol:

1. Heart rate response to deep breathing (E : I ratio).

2. Valsalva maneuver (Valsalva ratio).

3. 30 : 15 Ratio (lying to standing test).

4. Isometric handgrip test (IHT).

5. Cold pressor test (CPT).

6. Lying to standing SBP drop (ΔSBP).

Each test was scored and interpreted using:

1. Ewing’s classification.

2. Bellavere criteria.

3. AIIMS AFT Lab Grading System (for sympathetic and parasympathetic components).

Heart rate variability analysis

HRV was recorded via ECG using BIOPAC MP150 (BIOPAC Systems, Goleta, California, USA). Data were analyzed using Lab Chart 7 (AD Instruments, Dunedin, New Zealand).

Both time-domain and frequency-domain parameters were evaluated:

1. Time-domain: SD of all N–N intervals (SDNN), root mean square of all SDs (RMSSD), pNN50.

2. Frequency-domain: total power, low frequency, high frequency, low frequency/high frequency ratio.

Insulin resistance assessment

HbA1c was measured using an HPLC ion-exchange assay on the Bio-Rad D-10 system (NGSP-certified, IFCC-traceable; analytical range 4–18%; intra- and interassay coefficient of variations 1.1 and 1.6%). Fasting plasma glucose was estimated by the enzymatic hexokinase method on the Roche Cobas c311 analyzer (reportable range 20–600 mg/dl; analytical sensitivity 2 mg/dl; total CV < 2%). Serum insulin was measured using a chemiluminescent microparticle immunoassay on the Abbott Architect i2000SR platform (functional sensitivity 2µμIU/ml; intra- and interassay coefficient of variations 4.5 and 6.2%; proinsulin cross-reactivity < 0.5%).

Fasting insulin and plasma glucose levels were used to compute the HOMA-IR using the formula [18]:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{H}\mathrm{O}\mathrm{M}\mathrm{A}\text{-}\mathrm{I}\mathrm{R}}\\ =(\mathrm{F}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{i}\mathrm{n}\mu \mathrm{U}/\mathrm{m}\mathrm{l}\\ \times \mathrm{F}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{g}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{e}\mathrm{m}\mathrm{g}/\mathrm{d}\mathrm{l})/405.\end{array}}$$

Statistical analysis

Data were analyzed using SPSS version 21.0 (IBM Corp., USA). Continuous variables were assessed for normality using the Kolmogorov–Smirnov test. Depending on the distribution, comparisons between the two groups were made using either the unpaired t test for parametric data or the Mann–Whitney U test for nonparametric data. Categorical variables were compared using the χ² test. To examine associations between autonomic function parameters, HRV indices, insulin resistance, and hypoglycemia unawareness, correlation analyses were conducted using Pearson’s or Spearman’s correlation coefficients as appropriate. Multivariate regression analysis was performed to identify independent predictors of autonomic dysfunction. Statistical significance was defined as P < 0.05, with a confidence interval of 95% and statistical power set at 80% (β = 0.2).

Results

Participant characteristics

The study included 50 individuals with T2DM, divided into two groups: 25 individuals with hypoglycemia unawareness (hypoglycemia unawareness group) and 25 individuals without hypoglycemia unawareness (control group). Both groups were matched for age and sex. However, hypoglycemia unawareness group showed significantly higher BMI, SBP, DBP, and mean arterial pressure (all P < 0.05). No significant difference was observed in duration of diabetes, treatment modality, or resting heart rate (Table 1).

Table 1.

Baseline demographic and clinical characteristics of participants

Parameter	HU (mean ± SD)	Control (mean ± SD)	P value
Age (years)	56.20 ± 4.06	58.08 ± 4.11	0.11
Sex (M : F)	14 : 11	13 : 12
BMI (kg/m²)	27.3 ± 2.4	24.8 ± 2.1	0.012*
SBP (mmHg)	138.2 ± 10.4	129.1 ± 9.8	0.019*
DBP (mmHg)	86.6 ± 6.3	79.4 ± 7.1	0.007**
MAP (mmHg)	103.8 ± 5.1	96.0 ± 6.0	0.002**
Treatment (OHA/insulin)	Matched	Matched

DBP, diastolic blood pressure; HU, hypoglycemia unawareness; MAP, mean arterial pressure; OHA, oral hypoglycemic agents; SBP, systolic blood pressure.

^*P value < 0.05 Significant.

^**P value < 0.01 highly significant.

Patients with hypoglycemia unawareness were managed using regimens designed to minimize hypoglycemic risk. Drugs with high hypoglycemia potential, such as sulfonylureas and meglitinides, were avoided. Most patients received metformin with additional low-risk agents such as dipeptidyl peptidase-4 inhibitors, sodium–glucose co-transporter 2 inhibitors, or glucagon-like peptide-1 receptor agonists. Those requiring insulin were treated mainly with long-acting basal insulin analogues using cautious titration. Overall, therapy emphasized maintaining glycemic control while reducing glycemic variability and avoiding hypoglycemia.

Insulin resistance

Fasting blood glucose (FBG) levels were significantly higher in hypoglycemia unawareness group (P < 0.05); however, there was no significant difference in fasting insulin levels or HOMA-IR scores between the two groups (Table 2).

Table 2.

Comparison of fasting glucose, insulin, and homeostatic model of assessment – insulin resistance between groups

Parameter	HU (mean ± SD)	Control (mean ± SD)	P value
Fasting blood glucose (mg/dl)	114.36 ± 11.81	106.44 ± 13.11	0.021*
Serum insulin (µU/ml)	4.91 ± 2.67	4.75 ± 2.78	0.823
HOMA-IR	1.39 ± 0.75	1.23 ± 0.7	0.404

HOMA-IR, homeostatic model of assessment – insulin resistance; HU, hypoglycemia unawareness.

^*P value < 0.05 significant.

Autonomic function test results and heart rate variability analysis

Autonomic function testing revealed significantly greater dysfunction in the hypoglycemia-unaware group across both parasympathetic and sympathetic domains.

1. The Valsalva ratio was significantly lower in hypoglycemia unawareness group (1.49 ± 0.31) compared with the control group (1.80 ± 0.42) (P = 0.004).

• 2.CPT results demonstrated a significantly reduced rise in diastolic BP in hypoglycemia unawareness group (10.92 ± 9.81 mmHg) compared with the control group (18.36 ± 8.50 mmHg) (P = 0.001).
• 3.A greater fall in systolic BP during the lying-to-standing test (ΔSBP) was observed in hypoglycemia unawareness group (11.08 ± 6.38 mmHg) vs. the control group (7.64 ± 3.53 mmHg) (P = 0.037).

HRV parameters showed a significant reduction in global autonomic modulation in the hypoglycemia-unaware group.

• 1.SDNN, RMSSD, and pNN50 values were lower in hypoglycemia unawareness group, though only SDNN reached statistical significance.
• 2.Total power was significantly reduced in hypoglycemia unawareness group, indicating depressed overall autonomic modulation.
• 3.Low frequency was reduced, and low frequency/high frequency ratio was increased in hypoglycemia unawareness group compared with the control group, although the differences were not statistically significant, as shown in Table 3.

Table 3.

Comparison of autonomic function test parameters between groups

Test	Normal value	HU (mean ± SD)	Control (mean ± SD)	P value
E : I ratio	≥1.21	1.43 ± 0.15	1.45 ± 0.22	0.808
Valsalva ratio	≥1.21	1.49 ± 0.31	1.80 ± 0.42	0.004**
30 : 15 Ratio (LST)	≥1.04	1.25 ± 0.22	1.32 ± 0.17	0.066
ΔDBP in HGT (mmHg)	≥16	17.60 ± 8.62	18.00 ± 6.33	0.852
ΔDBP in CPT (mmHg)	≥16	10.92 ± 9.81	18.36 ± 8.50	0.001**
ΔSBP in LST (mmHg)	≤10	11.08 ± 6.38	7.64 ± 3.53	0.037*
Time-domain parameters
SDNN (ms)		45.07 ± 9.92	52.86 ± 13.97	0.084
RMSSD (ms)		39.92 ± 12.89	45.84 ± 11.68	0.060
pNN50 (%)		19.01 ± 15.08	24.89 ± 13.83	0.052
Frequency-domain parameters
Total power (ms²)		2001.56 ± 971.52	3015.03 ± 2108.67	0.052
LF (ms²)		534.39 ± 417.42	566.61 ± 328.47	0.207
HF (ms²)		627.9 ± 445.65	775.35 ± 360.87	0.057
LF/HF ratio		1.06 ± 0.65	0.86 ± 0.55	0.277

CPT, cold pressor test; DBP, diastolic blood pressure; E : I ratio, expiratory to inspiratory ratio; HF, high frequency; HGT, hand grip test; HU, hypoglycemia unawareness; LF, low frequency; LST, lying to standing test; pNN50%, percentage of N–N intervals differing by 50 ms from the previous one; RMSSD, root mean square of all SDs; SBP, systolic blood pressure; SDNN, SD of all N–N intervals.

^*P value < 0.05 significant.

^**P value < 0.01 highly significant.

Classification of autonomic dysfunction

Assessment using standard scoring systems revealed a higher burden of autonomic neuropathy in the hypoglycemia-unaware group.

• 1.AIIMS AFT Lab scoring showed significantly greater sympathetic dysfunction in hypoglycemia unawareness group (1.64 ± 0.86) vs. the control group (2.44 ± 0.92) (P = 0.003), and greater parasympathetic impairment (2.72 ± 0.68 vs. 3.00 ± 0.00; P = 0.039) (Table 4).

Table 4.

Grading of autonomic dysfunction based on All India Institute of Medical Sciences autonomic function test lab criteria (parasympathetic and sympathetic) and Bellavere criteria

Scoring system	HU (mean ± SD)	Control (mean ± SD)	P value
Bellavere’s score	0.64 ± 1.08	0.00 ± 0.00	0.001***
AIIMS sympathetic score	1.64 ± 0.86	2.44 ± 0.92	0.003**
AIIMS parasympathetic score	2.72 ± 0.68	3.00 ± 0.00	0.039*

AIIMS, All India Institute of Medical Sciences; HU, hypoglycemia unawareness.

^*P value < 0.05 significant.

^**P value < 0.01 highly significant.

^***P value < 0.001 very highly significant.

A larger proportion of subjects with hypoglycemia unawareness were classified as having ‘early’ or ‘definite’ CAN as described by AIIMS AFT Lab criteria and Bellavere criteria (Fig. 2).

Fig. 2.

Categorization of cardiac autonomic neuropathy in type 2 diabetes mellitus individuals with and without hypoglycemia unawareness using AIIMS AFT Lab scores and Bellavere’s criteria (left: sympathetic domain; right: parasympathetic domain). AFT, autonomic function test; AIIMS, All India Institute of Medical Sciences.

Correlation analysis

Pearson and Spearman correlation analyses revealed:

• 1.A negative correlation between serum insulin levels and sympathetic function indices, such as CPT, IHT, and ΔSBP.
• 2.A significant negative correlation between SDNN, IHT, and Bellavere score, indicating that greater autonomic dysfunction was associated with lower HRV, as shown in Table 5.

Table 5.

Correlation between autonomic function/heart rate variability and homeostatic model of assessment – insulin resistance

Parameter	Correlation with HOMA-IR	Correlation coefficient (r/ρ)	P value	Correlation with Bellavere score	Correlation coefficient (r/ρ)	P value
ΔSBP (lying-to-standing test)	Negative	−0.253	0.173	Positive	0.301	0.144
Cold pressor test (ΔDBP)	Negative	−0.146	0.496	Negative	−0.032	0.880
Isometric handgrip test	Negative	−0.319	0.104	Negative	−0.428	0.033*
SDNN	Positive	0.025	0.548	Negative	−0.422	0.036*

DBP, diastolic blood pressure; HOMA-IR, homeostatic model of assessment – insulin resistance; SBP, systolic blood pressure; SDNN, SD of all N–N intervals.

^*P value < 0.05 significant.

Discussion

Overview of key findings

This study aimed to assess the relationship between hypoglycemia unawareness, insulin resistance, and autonomic function in individuals with T2DM. Our findings demonstrated that individuals with hypoglycemia unawareness had significantly greater autonomic dysfunction, affecting both sympathetic and parasympathetic domains, compared to those without hypoglycemia unawareness. The Valsalva ratio, CPT, and lying-to-standing systolic BP response (ΔSBP) were significantly impaired in the hypoglycemia-unawareness group. Grading by Ewing’s and Bellavere criteria, as well as AIIMS AFT scoring, confirmed a higher burden of CAN in this group. HRV indices, especially SDNN and total power, were significantly reduced, indicating impaired sympatho-vagal modulation. While FBG levels were higher in the hypoglycemia unawareness group, there was no significant difference in insulin levels or HOMA-IR scores between the groups. However, serum insulin showed a negative correlation with sympathetic function parameters, suggesting a possible link between insulin resistance and sympathetic impairment.

Autonomic impairment in the individuals

Our results are in line with previous studies demonstrating a strong association between autonomic dysfunction and hypoglycemia unawareness in diabetic individuals [9,19–23]. Cryer et al. [9] described the phenomenon of HAAF, where recurrent hypoglycemia attenuates the autonomic counter-regulatory response, thereby increasing the risk of future severe hypoglycemic events. Similarly, studies by Heller et al. [19] and Freeman and Chapleau [20] have shown that reduced sympathoadrenal activity, particularly in those with long-standing diabetes or on insulin therapy, leads to blunted adrenergic symptoms and impaired glucose counter-regulation. Our findings are also supported by Cichosz et al. [21], who found that altered HRV responses during hypoglycemia were often confounded by coexisting CAN. A previous study by Adeva-Andany et al. [22] suggested that hyperinsulinemia is associated with increased cardiovascular autonomic dysfunction. Schoot et al. [23] also showed that aggressive treatment of hyperglycemia was also shown to play a role in Hypoglycemia unawareness and insulin resistance. This is in alignment with our study showed that mean serum insulin concentrations in the hypoglycemia-unawareness T2DM individuals were higher than in the control group.

Comparison between autonomic function tests and hypoglycemia unawareness

The AFTs in our study highlight both parasympathetic and sympathetic deficits in individuals with hypoglycemia unawareness. The lower Valsalva ratio on the Valsalva maneuver test, an exaggerated drop in systolic BP during postural change, indicates impaired baroreflex sensitivity and vagal tone as shown by previous studies [24,25]. The difference between the two groups was highly significant (P = 0.017), as seen in the study by Bonette et al. [26] in diabetic individuals age- and sex-matched controls, while in the study by Kruter et al. [27], an inverse relationship was shown between Valsalva ratio and the prevalence of retinopathy and nephropathy and the duration of diabetes mellitus. Low Valsalva ratio values correlated well with the prevalence and severity of complications.

A reduced rise in diastolic BP during CPT and blunted response during IHT point toward impaired sympathetic reactivity, also shown by Bond et al. [28]. In the deep breathing test the E : I ratio and ∆HR were in the normal range in both groups, but the values were decreased in the hypoglycemia unawareness group as compared with the control group. A study by Brown et al. [29] in T2DM subjects showed that the E : I ratio was significantly reduced. Sridhar et al. [30] also found that the R–R interval variation obtained during the deep breath test was much lower in the people with diabetes group than in normal controls.

In our study, both the hypoglycemia unawareness group and the control group showed deranged values following the handgrip dynamometer test and the mean rise in DBP in case of hypoglycemia unaware group (mean = 17.60 ± 8.62 mmHg) was less as compared with that in control group (mean = 18.00 ± 6.33 mmHg), though this was not statistically significant (P = 0.852), as seen in study by Kunutsor et al. [31]. Although a study by Anna et al. [32] showed that the handgrip test should no longer be part of the cardiovascular autonomic testing, being highly dependent on hypertensive status and baseline diastolic BP.

In our study, the mean of the case group showed borderline values (11.08 ± 6.38 mmHg), whereas the mean value in the control group (7.64 ± 3.53 mmHg) was within normal limits. This difference between the case group and control group showed statistical significance (P = 0.037). This finding is in line with respect to the study by Fedorowski and Gibbons [33], where T2DM individuals showed a significant difference from matched controls in ∆SBP on lying to standing.

Insulin resistance and clinical implications

Although no significant difference in HOMA-IR was observed, the negative correlations between Bellavere score and autonomic parameters suggest that hyperinsulinemia may contribute to sympathetic dysfunction, as also seen by Frontoni et al. [34] and Ramesh et al. [35]. Chronic exposure to elevated insulin has been linked to endothelial dysfunction, increased sympathetic tone, and reduced baroreflex sensitivity, all of which may contribute to the development of CAN [36–38]. These findings highlight the need for early recognition of subtle autonomic changes even in the absence of overt hypoglycemia symptoms or elevated HOMA-IR.

These findings also underscore the importance of routine autonomic screening in individuals with T2DM, particularly those with long-standing disease or insulin therapy. The HRV results further support these observations, as significantly reduced SDNN and total power in the hypoglycemia unawareness group reflect diminished autonomic adaptability and an overall decrease in sympatho-vagal modulation. Chronic hyperglycemia, recurrent hypoglycemia, and possible hyperinsulinemia drive autonomic dysfunction, leading to hypoglycemia unawareness, cardiovascular risk, and a self-perpetuating cycle of HAAF.

Strengths and limitations

A major strength of this study is the use of comprehensive autonomic testing protocols, validated scoring systems (Bellavere and AIIMS AFT Lab criteria), and HRV analysis, providing both qualitative and quantitative assessment of autonomic function. The inclusion of age- and sex-matched groups, and the exclusion of confounding comorbidities, enhanced internal validity. In addition, the use of Clarke’s questionnaire allowed for standardized detection of hypoglycemia unawareness.

However, this study has some limitations. The relatively small sample size may have limited the power to detect subtle associations, particularly for insulin resistance and HRV frequency components. Second, insulin resistance was measured using HOMA-IR, which, although widely used, may not fully reflect dynamic insulin sensitivity. Also, the people recruited in this study may not necessarily be representative of the broader population of people with type 2 diabetes. Lastly, the influence of antecedent hypoglycemic episodes could not be objectively measured, which may have contributed to variability in autonomic responses.

Conclusion

In conclusion, our study demonstrates a clear association between hypoglycemia unawareness and cardiac autonomic dysfunction in individuals with T2DM. The presence of both sympathetic and parasympathetic impairments, along with reduced HRV parameters, suggests that hypoglycemia unawareness may be both a marker and consequence of underlying CAN. While insulin resistance did not significantly differ between the groups, its correlation with autonomic dysfunction warrants further investigation. These findings underscore the importance of early autonomic function screening in diabetic individuals, particularly those with recurrent or unrecognized hypoglycemia. Incorporating autonomic assessment into routine diabetes care may aid in risk stratification and prevention of adverse cardiovascular outcomes.

Acknowledgements

D.N., Su.G. designed the study, interpreted the results, drafted the manuscript were involved in the clinical care of the individuals, and accessed and verified the underlying data. D.N. and A.T. collected the data, reviewed, and edited the manuscript. Sa.G., S.Z., and R.S. reviewed and edited the manuscript and were involved in the clinical care of the individuals. Sa.G. and N.A. helped in the recruitment of the subjects and reviewed and edited the manuscript. All the authors accept responsibility for the decision to submit this manuscript for publication.

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of interest

There are no conflicts of interest.