Nasal versus Buccal Breathing and Non-Invasive Indices of Vascular Function in Healthy Young Adults: An Observational Crossover Study

Introduction

Breathing is a fundamental physiological process that influences multiple systems, including cardiovascular regulation, acid–base balance, and physical performance. Respiratory patterns interact with autonomic regulation, cardiovascular control, and vascular tone, thereby influencing peripheral hemodynamics. It is also modulated by psychological and emotional states and is closely linked to cardiac function.

Nasal versus oral (buccal) breathing has become a topic of interest in respiratory research, with growing evidence of its impact on cardiovascular physiology. The paranasal sinuses produce nitric oxide (NO) at concentrations several times higher than those in the alveoli.^1–4 This NO is continuously synthesized by the ciliated epithelium via inducible nitric oxide synthase (iNOS) and plays key roles in airway defense, mucociliary clearance, and pulmonary perfusion.^5–7

Clinically, nasal NO is used as a biomarker in respiratory diseases: levels are increased in inflammatory airway conditions and reduced in primary ciliary dyskinesia. Beyond respiratory function, NO exerts potent vasodilatory effects, particularly during physical activity, where shear stress stimulates endothelial NO release, promoting arterial relaxation.^8,9 Recent evidence further indicates that nasal breathing, compared with oral breathing, acutely lowers diastolic blood pressure and increases parasympathetic contributions to heart rate variability in healthy young adults.¹⁰ Existing studies examining breathing interventions have largely relied on exercise paradigms, paced or deep breathing techniques, or patient populations, making it difficult to isolate the specific contribution of breathing route itself to vascular responses under calm, resting conditions.¹¹

Despite these known mechanisms, the specific influence of nasal versus buccal breathing on vascular stiffness remains poorly characterized. Arterial stiffness and endothelial responsiveness are key determinants of cardiovascular health. Understanding this relationship may provide insight into physiological mechanisms relevant to cardiovascular regulation, a condition strongly associated with impaired endothelial NO bioavailability.^10,11 Whether acute changes in breathing route are associated with measurable differences in these indices under resting physiological conditions has not been well established.

Nasal breathing represents the physiological mode of respiration. However, in the presence of nasal obstruction—such as a deviated nasal septum, chronic rhinosinusitis, or persistent allergic conditions—individuals may rely more heavily on buccal breathing. While breathing patterns are known to influence cardiovascular regulation, most previous studies have focused on respiratory mechanics, autonomic modulation, or gas exchange, rather than on peripheral vascular properties. In particular, the acute effects of breathing route (nasal versus buccal) on non-invasive indices of vascular elasticity and arterial function have received little direct investigation.

The aim of this study was therefore to compare the effects of nasal and buccal breathing on non-invasive indices of vascular function, using two validated non-invasive methods: digital plethysmography (DP) and ultrasound flow-mediated dilation (US-FMD) in healthy young adults. By elucidating the vascular effects of different breathing routes, we sought to highlight their potential clinical relevance for cardiovascular health and hypertension management.

Materials and Methods

Study Design and Ethical Approval

This research was an observational crossover physiological study without intervention, therefore it was not registered as a clinical trial. The protocol was approved by the Academic Bioethics Committee (approval number B200-2018-111) and complied with the principles of the Declaration of Helsinki. All participants provided written informed consent prior to participation. The study procedures are described in detail in a publicly available methods paper, which provides the methodological framework for the present study.¹²

This study was designed to investigate acute, short-term vascular responses to different breathing routes within a single experimental session. Accordingly, the protocol was not intended to assess long-term adaptations, and all outcomes are interpreted as immediate, condition-specific effects observed under controlled laboratory conditions.

All experimental sessions were conducted in a quiet, temperature-controlled indoor laboratory environment. Measurements were performed at similar times of day to minimize circadian variability.

Participants

Forty-five healthy Caucasian male volunteers (mean age 21.3 ± 2.3 years) were recruited. The peripheral vascular function was verified using the Ankle–Brachial Index (ABI 1.0 ± 0.06), calculated as the ratio of systolic blood pressure in the lower versus upper limbs, a recognized screening marker of atherosclerosis and predictor of cardiovascular risk.^12,13

Inclusion criteria required that participants be non-smokers, refrain from alcohol and caffeine for 24 hours, free of cardiovascular disease, narcotics and drugs, sleep disorders, or prior respiratory surgery. Hydration status was verified on arrival using standardized pretest instructions (1 liter water 12 hours before and 1 liter water prior to the test session). Volunteers were asked to refrain from consuming dark chocolate and coffee before testing due to its known effects on flow-mediated dilation.¹⁴

Breathing Protocols

Each subject completed two breathing protocols—nasal breathing (NB) and buccal breathing (BB)—in randomized order. In both protocols, breathing was performed spontaneously under calm, resting conditions, without any instruction to alter breathing depth or pattern (ie. no deep or paced breathing). Protocols were separated by a standardized washout period consisting of a 10-minute gentle walk followed by a 30-minute seated rest period to minimize carryover effects. During the BB protocol, a nose clip ensured exclusive oral breathing. (Figure 1)

Figure 1 Randomly followed procedure by the healthy participants. The red coloured circle represents the trajectory followed by the subject. Abbreviations: N, number of participants; NB, nasal breathing; BB, buccal breathing.

Assessment of Non-Invasive Indices of Vascular Function

Digital Plethysmography (DP)

Peripheral vascular endothelial function was evaluated using digital plethysmography (Iworx IX-214 4-Ch Data Recorder and pulse transducer PT 100, Dover NH, USA).^15,16 The sensor was placed on the index finger, and pulse waveforms were recorded under standardized conditions. From these recordings, two indices were derived:

• Pulse Propagation Time (PPT): the interval between the systolic and dicrotic pulses, reflecting smooth muscle relaxation. (Figure 2)
• Reflection Index (RI): the ratio of the dicrotic pulse amplitude to the systolic pulse amplitude (B/A), representing arterial stiffness. (Figure 2)

Figure 2 Illustration of digital plethysmography trace on the index finger. Pulse propagation time (PPT) = time between the systolic and the dicrotic pulse. Reflection Index (RI) = (B/A), where A = systolic pulse and B = dicrotic pulse.

The PPT index reflects smooth muscle relaxation of the artery, and RI represents a waveform-derived index related to arterial tone and pulse wave reflection.^17,18

Ultrasound Flow-Mediated Dilation (US-FMD)

Endothelial function was also assessed by ultrasound-based flow-mediated dilation of the brachial artery, performed in accordance with consensus guidelines and protocol of Thijssen et al, 2019.¹⁹ Brachial arterial diameter was measured at rest and after each breathing protocol. Two experienced radiologists independently analyzed the ultrasound images. (Figure 3)

Figure 3 Brachial artery dilation was measured after application of the flow-mediated dilation protocol.30 Yellow arrow: diameter of the brachial artery. (A) control measure at rest; (B) after the nasal breathing (NB) or buccal breathing (BB) protocol.

Both US-FMD and DP can significantly screen cardiovascular events, making them good prognostic markers.²⁰

Statistical Analysis

Normality of data distribution was assessed using the Shapiro–Wilk test. Changes within each protocol were analyzed using the repeated-measures Friedman test. Differences between the NB and BB protocols were evaluated using the Wilcoxon signed rank test or paired t-test, as appropriate. A two-sided p value < 0.05 was considered statistically significant. A post-hoc sensitivity power analysis was performed to assess the detectable effect size given the final sample size. With 45 participants in a within-subject crossover design and a two-sided significance level of α = 0.05, the study had 80% power to detect a standardized mean paired difference of approximately Cohen’s dz = 0.43, indicating sufficient sensitivity to identify small-to-moderate differences between breathing conditions.

Results

Participant Characteristics

Participant characteristics were summarized at baseline. All participants were young, healthy male adults with normal peripheral vascular function, as confirmed by Ankle–Brachial Index values within the normal range. No participant had a history of cardiovascular, respiratory, or metabolic disease, and all measurements were performed under standardized resting conditions.

Plethysmography

Nasal breathing significantly increased pulse propagation time (PPT) compared with baseline, indicating changes consistent with increased vascular relaxation. This effect persisted through the third measurement and was absent during buccal breathing.

Direct comparison between breathing protocols demonstrated consistently higher PPT values (6.35% ± 1.51) during nasal breathing than buccal breathing (p < 0.001), indicating lower stiffness-related index values during nasal breathing. In addition, the Reflection Index (RI) was significantly higher (4.6% ± 1.25) during buccal breathing compared with nasal breathing (p = 0.024), reflecting differences in stiffness-related indices. Similar trends were observed at measurement 3, with significant variations persisting for both PPT (p < 0.001) and RI (p < 0.05) (Figure 4).

Figure 4 Evolution of the plethysmography trace recorded on the index finger. * p< 0.05; *** p<0.001. Abbreviations: NB, Nasal Breathing; BB, Buccal Breathing; PPT, Point to point time (time between the systolic and the dicrotic pulse); RI, Reflection Index; ns, non-significant.

Flow-Mediated Dilation

Ultrasound flow-mediated dilation revealed a significant increase in brachial artery diameter (4.46% ± 0.05) during the nasal breathing protocol (Measure 2 vs. 1: p < 0.001; Measure 3 vs. 2: p < 0.001; Measure 3 vs. 1: p < 0.001). No such changes were observed during buccal breathing (Figure 5).

Figure 5 Evolution of the diameter of the brachial artery. ** p<0.01; *** p<0.001. Abbreviations: NB, Nasal Breathing; ns, non-significant.

Direct comparison of the two protocols demonstrated that nasal breathing elicited significantly greater brachial artery dilation (Measure 2: p < 0.001; Measure 3: p < 0.01). These results indicate that nasal breathing induces a measurable increase in brachial artery diameter, whereas oral breathing does not produce a statistically significant change (Figure 6).

Figure 6 Increased brachial artery diameter evaluated by ultrasound flow-mediated dilation protocol between nasal and buccal breathing. ** p<0.01; *** p<0.001. Abbreviations: NB, Nasal Breathing; BB, Buccal Breathing.

Discussion

To our knowledge, this study is among the first to demonstrate that changing breathing route alone, without paced respiration or exercise, is associated with detectable differences in non-invasive indices of vascular function in a healthy population. This study demonstrates that nasal breathing is associated with greater peripheral arterial dilation and differences in non-invasive indices of vascular function compared with buccal breathing. One potential mechanism discussed in the literature involves nasal nitric oxide; however, NO levels were not directly measured in this study, and its role therefore remains speculative. Importantly, these findings should be interpreted within the physiological context of a young, healthy population. Participants did not exhibit elevated arterial stiffness or endothelial dysfunction at baseline.

Blood pressure and heart rate were not measured during the experimental protocols, which limits mechanistic interpretation and direct translation of the observed vascular responses. However, as this was an observational crossover study focusing on within-subject comparisons, results were interpreted as relative differences in vascular indices between breathing conditions under standardized experimental conditions, rather than as definitive evidence of underlying hemodynamic or autonomic mechanisms.

Although acute physical activity such as walking may transiently influence vascular indices, the washout procedure—including the gentle walk and subsequent seated rest—was identical for all participants and conditions. Therefore, outcomes were interpreted as condition-specific differences assessed under this standardized protocol rather than as absolute resting values.

Hypertension is a multifactorial condition and a leading risk factor for cardiovascular disease. It induces structural and functional changes in arteries, including impaired endothelial vasodilation and reduced NO synthesis.^21–23 A decline in NO bioavailability contributes to the pathophysiology of hypertension and is also associated with pulmonary hypertension, insulin resistance, erectile dysfunction, and preeclampsia. Increasing evidence suggests that an imbalance between NO production and endothelin-1 may contribute to blood pressure regulation, even in children and adolescents.^24–27

Nitric oxide has been proposed in the literature as a potential mediator of vascular responses; however, it was not measured in the present study, and its involvement cannot be inferred from these data. Further studies incorporating direct physiological measurements are needed to clarify underlying mechanisms and their relevance in populations at cardiovascular risk.²⁸

The results also align with growing evidence that waveform analysis provides valuable prognostic information beyond central blood pressure, such as pulse wave reflection effects. By promoting vasodilation and reducing arterial stiffness, nasal breathing may represent a simple, nonpharmacological strategy to support vascular health.²⁹

Limitations

This study has several limitations that should be considered when interpreting the findings.

First, this was an acute, observational crossover study conducted under controlled laboratory conditions, and the observed differences represent short-term, within-subject responses to breathing route rather than long-term physiological adaptations. Consequently, the results cannot be extrapolated to chronic breathing patterns or to clinical outcomes.

Second, the study population consisted exclusively of young, healthy male adults with normal peripheral vascular function at baseline. As such, participants did not exhibit elevated arterial stiffness or endothelial dysfunction, and the findings should be interpreted as differences in non-invasive vascular indices and vasodilatory responses rather than reductions of pathological vascular stiffness. Generalization to females, older individuals, or populations with cardiovascular or respiratory disease should therefore be made with caution.

Third, key hemodynamic and autonomic variables, including blood pressure and heart rate, were not measured during the experimental protocols. While this limits mechanistic interpretation of the observed vascular responses, the crossover design and standardized experimental conditions allow for valid relative comparisons between breathing routes within individuals.

Fourth, nitric oxide was not directly measured. Although nasal nitric oxide has been proposed in the literature as a potential mediator of vascular responses to breathing route, its involvement cannot be inferred from the present data and remains speculative.

Fifth, breathing was performed spontaneously without control of respiratory depth or pattern. While this approach was chosen to reflect natural resting breathing, it may have introduced inter-individual variability in ventilatory parameters that could influence vascular responses.

Finally, although ultrasound images were independently reviewed by two experienced radiologists and measurements were averaged for analysis, formal assessment of inter-observer reliability was not performed, which may have introduced measurement variability.

Conclusion

The present study examined short-term vascular responses to different breathing routes under standardized conditions. Nasal and buccal breathing were associated with distinct patterns in non-invasive indices of vascular function in healthy young adults.

Future research should extend these observations to individuals with habitual mouth breathing to determine whether chronic breathing patterns are associated with sustained differences in vascular function. Studies incorporating direct physiological measurements are needed to clarify the mechanisms underlying these vascular responses.