Abstract Menopause accelerates declines in vascular function and physical performance, particularly among women with elevated blood pressure. Interventions that can simultaneously target both vascular and functional domains offer important clinical opportunities. This study investigated the efficacy of a 4-week square-stepping exercise program on arterial stiffness, balance, and endurance in postmenopausal women with elevated blood pressure or stage 1 hypertension. Thirty-two postmenopausal women (mean age 53.8 ± 2.3 years) were randomly assigned to either a square-stepping exercise group (n = 16) or a non-exercise control group (n = 16). Data were analyzed using paired t-tests, independent samples t-tests, and Pearson correlations coefficients (P < 0.05). The exercise group performed moderate intensity square-step exercise for 40 minutes/session, 5 days/week, for 4 weeks. Arterial stiffness (brachial-ankle pulse wave velocity, baPWV), dynamic balance (Timed Up and Go test), and functional endurance (2-minute step test) were assessed at baseline and post-intervention. After 4 weeks, the square-stepping exercise group demonstrated a significant improvement in arterial function, indicated by a reduction in baPWV (Δ = -0.91 m/s, P < 0.05). Balance and cardiorespiratory endurance also improved significantly in the exercise group (P < 0.05). Correlation analyses revealed that the magnitude of baPWV reduction was positively associated with balance improvements (r = 0.61, P < 0.001) and also related with endurance gains (r = -0.46, P < 0.01). A short-term square-stepping program represents an effective intervention for concurrently enhancing vascular function, balance, and endurance in postmenopausal women with elevated blood pressure. This approach may reduce cardiovascular risk and promote functional independence in this population.

Keywords: Menopause, Physical performance, Pulse wave velocity, Square exercise, Vascular function

Funding: This study was supported by “Research and Training Center for Enhancing Quality of Life of Working-Age People” and “Research and Graduate Studies” Khon Kaen University.

Citation:  Nakmareong, S., Thasomboon, M., Wongsaya, E., Pimdee, A., and Adchaithor, P. 2026. Regular square-stepping exercise promotes arterial function in postmenopausal women. Natural and Life Sciences Communications. 25(3): e2026067.

Graphical Abstract:

INTRODUCTION

Menopause marks a period of elevated cardiovascular disease risk for women and contributes to increased rates of both cardiovascular-related and all-cause mortality (Muka et al., 2016). A hallmark of this transition is the precipitous decline in the estrogen hormone, which has well-established cardioprotective effects. The resulting hypoestrogenic state accelerates cardiovascular ageing through multiple pathways, including metabolic dysregulation and adverse hemodynamic change (Nair et al., 2021).

Arterial stiffness, characterized by a loss of vascular elasticity, is an early marker of vascular ageing and a key contributor to hypertension. This condition can lead to adverse cardiovascular outcomes, including ventricular hypertrophy and impaired coronary perfusion (Chirinos et al., 2019). Large cohort studies have demonstrated an association between menopause and increased arterial stiffness (Vallee, 2025). Furthermore, evidence suggests that menopause modulates the progression of age-associated vascular impairment, suggesting that hormone changes as key regulators of vascular function (Takahashi et al., 2005). Therefore, implementing strategies to mitigate cardiovascular events is essential for this at-risk population.

In addition to cardiovascular concerns, postmenopausal women often experience declines in physical function, including balance deficits and reduced cardiorespiratory fitness (Zhao et al., 2020; Bondarev et al., 2021). This combination of health challenges diminishes quality of life and increases the risk of disability, contributing significantly to frailty and the consequent loss of personal independence (Ruan et al., 2020). In this context, exercise emerges as a potent, nonpharmacological strategy to address these functional declines and improve health outcomes in this population.

A square-stepping exercise is a functional training method that involves structured, multidirectional stepping sequences performed on a square grid. This intervention is simple to implement, cost-effective, and requires no specific equipment or setting. Previous studies have shown that square-stepping exercise programs lead to significant improvements in health parameters such as agility, postural stability, strength, and aerobic endurance (Fisseha et al., 2017; Siqueira et al., 2024). Additionally, stepping exercise enhances lower‑limb muscle strength, promotes endothelial shear stress and augments muscle-pump action, all of which may attenuate arterial stiffness (Ohta et al., 2012; Manojlovic et al., 2021). However, despite the established benefits for physical and cognitive function, the effect of square-stepping exercises on arterial stiffness in postmenopausal women remains unclear Therefore, this study aimed to investigate the effects of a four-week square-stepping exercise intervention on arterial stiffness, balance, and functional capacity in middle-aged postmenopausal women with elevated blood pressure or stage 1 hypertension. We hypothesized that a 4-week moderate-intensity square-stepping exercise program would reduce arterial stiffness, improve balance and enhance functional endurance in postmenopausal women with elevated blood pressure or stage 1 hypertension, with improvements in vascular function positively correlated with gains in balance and endurance.

MATERIALS AND METHODS

Study Setting and Subjects

The study protocol was review and approved by the University Ethics Committee in Human Research. All subjects have provided written consent in accordance with the Declaration of Helsinki prior to data collection. This study has been registered in the Clinical Trials Registry provided by the Thai Clinical Trails Registry.

Sample size

The sample size was determined based on an expected between-group difference in baPWV. Data from a previous exercise intervention study by Wong et al. (2018) indicated a mean reduction of 0.9 m/s in the exercise group versus 0.1 m/s in controls, yielding a between-group difference of 0.8 m/s. Using a Cohen’s d of 0.81 derived from pooled standard deviations reported in similar populations, a sample size of 14 per group was required to achieve 80% power at α = 0.05. Accounting for up to 15% attrition, we targeted 16 participants per group.

Participants

Middle-aged postmenopausal women (aged 40–59 years) with elevated blood pressure or stage 1 hypertension were recruited from communities around Khon Kaen University, Khon Kaen province, Thailand. Recruitment occurred via posters displayed at local Sub-district Health Promoting Hospitals. A flow chart of the study enrolment is demonstrated in Figure 1.

The inclusion criteria were: 1) women aged 40–59 years; 2) postmenopausal status, defined as the absence of spontaneous menses for ≥ 12 consecutive months not attribution to pregnancy, medical causes or surgically induced menopause and 3) elevated blood pressure or stage 1 hypertension, as defined by the American College of Cardiology and American Heart Association guidelines (Whelton et al., 2018), resting systolic blood pressure 120-139 mmHg or diastolic blood pressure 60-89 mmHg. Exclusion criteria included the following: 1) regular exercise (>150 minutes/week); 2) body mass index ≥ 30kg/m²; 3) use of antihypertensive medications or any other drugs that could affect the outcome variables; 4) use of hormone replacement therapy within the six months preceding the study; 5) a history of a fracture in the lower extremity or spine within the past six months; and 6) a history of cardiovascular, pulmonary, neurological, or musculoskeletal disorders that would contraindicate participation in the exercise program or interfere with testing procedures.

Participants were randomly allocated to either the square-stepping exercise group or the control group using a computer-generated randomization sequence.

Figure 1. Flow of participants in the study.

Square-stepping exercises

The square-stepping exercise intervention was adapted from the protocols in a previous study (Junprasert et al., 2013; Wannapong et al., 2021; Krabuanpatana, 2023) Prior to the main intervention, participants completed a one-week familiarization period (run-in phase). During this phase, a physiotherapist instructed participants on the square-stepping movement patterns until they could perform all movements accurately and independently. This phase also familiarized participants with intensity requirement, ensuring participant could archive and maintain target hear rate.

Participants in the intervention group underwent a four-week supervised training program consisting of 20 sessions conducted five times per week. Each session lasted 40 minutes and included a 5-minute warm-up, 30-minute continuous main exercise, and a 5-minute cool-down with stretching. The exercise protocol featured six distinct movement patterns (side-walking, diamond-shape, cross-shape, V-shape, oblique cross-leg, and oblique star radiance; Figure 2) performed on a standardized 3 × 3 grid comprising nine 25 cm × 25 cm squares (Krabuanpatana, 2023). Exercise intensity was prescribed at 60%–80% of each participant’s heart rate reserve, calculated using the Karvonen formula. Exercise intensity was controlled by continuously monitored heart rate throughout each session using a POLAR heart rate monitor (Polar Electro, Kempele, Finland). Real-time heart rate feedback enabled stepping speed adjustment during square-stepping exercise when heart rate fell below or exceed the target range, ensuring adherence to prescribed intensity.

Figure 2. Square-stepping movement patterns; side-walking (A), diamond-shape (B), cross-shape(C), V-shape (D), oblique cross-leg (E), and oblique star radiance (F).

Control group

Participants in the control group were instructed to continue their habitual lifestyle throughout the study period.

Outcome measures

The primary outcome measure was brachial-ankle pulse wave velocity (baPWV).  Secondary outcome measures included balance and aerobic capacity. All outcomes were assessed at baseline and 4-week post-exercise intervention by research assistant who was blinded to participant’s group allocation throughout the study. Prior to the study, the assessor completed a standardized training in all measurement protocols involving a pilot sample of 10 middle-aged women not part of the current study. The intra-class correlation coefficient for baPWV was 0.94. Similar high reliability was observed for balance and functional capacity tests, with ICC ranging from 0.90-0.92.

Pulse wave velocity

baPWV, a measure of arterial stiffness, was assessed using a data acquisition system (Arterial Compliance Monitor; Barts and The London School of Medicine and Dentistry, UK) with Doppler probes (Dopplex MDII; Huntleigh Healthcare, UK) (Usupharach et al., 2025). Pulse wave transit time was calculated as the time delay between the feet of the simultaneously recorded brachial and ankle waveforms. The distance between measurement sites was estimated from each participant’s height using a validated equation (Munakata, 2016). The baPWV value for each participant was the average of three separate measurements, with each measurement derived from 10 consecutive stable cycles.

To ensure standardized conditions, all assessments were conducted between 8:00 and 12:00 in a quiet, temperature-controlled room. Participants were instructed to abstain from vigorous exercise for 24 hours and from caffeine, alcohol, and nicotine for 12 hours before their appointment. Measurements were taken after a minimum 10-minute rest period in the supine position. To minimize the influence of circadian variation, each participant's post-intervention assessments were scheduled at the same time of day as their baseline visit (Laurent et al., 2006).

Balance

Functional balance was assessed using the Timed Up and Go (TUG) test. The procedure began with participants seated in a standard armless chair. They were instructed to stand, walk 3 meters at a self-selected comfortable pace, turn around a cone, return to the chair, and subsequently sit down. After one practice trial for familiarization, two test trials were completed. The average of the two trial times was recorded and used for analysis (Benavent-Caballer et al., 2016).

Functional capacity

Cardiorespiratory fitness was assessed using the two-minute step test. Before testing, the target height for each participant was marked on the wall at the midpoint between the patella and the anterior superior iliac crest. Participants were instructed to march in place for two minutes, lifting each knee to the target height. The test score was the total number of times the right knee reached the marked height within two minutes. This test was administered according to standardized protocols (Rikli et al., 1999).

Statistical analysis

Data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and are reported as means ± standard deviation (SD) unless otherwise specified. Statistical significance was set at P < 0.05. The normality of data distribution was verified using the Kolmogorov–Smirnov test.

Baseline demographic and clinical characteristics were compared between the square-stepping exercise and control groups using independent t-tests.

To evaluating the 4-week exercise intervention effect, within-group changes for baPWV, balance indices, and functional capacity were assessed using paired-samples t-tests. Subsequent between-group comparisons of these variables were conducted using independent-samples t-tests on the post-intervention values.

To examine the relationship between vascular and functional adaptations, Pearson correlation coefficients were calculated to determine the associations between changes (Δ post–pre) in baPWV and changes in the timed up and go and 2-minute step test performance.

RESULTS

Baseline demographic and clinical characteristics were comparable between the square-stepping exercise and control groups (P > 0.05; Table 1). For the total cohort, the mean age was 53.84 ± 2.29 years, mean systolic and diastolic blood pressures were 129.16 ± 5.50 mmHg and 73.72 ± 6.76 mmHg, respectively. The compliance rate with the exercise was more than 85%. There were no adverse effects over the course of training.

Table 1. Baseline characteristic of participants.

Note: presented as mean ± standard deviation. Abbreviation: BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate, bpm; beats per minute. P-values were obtained using independent t-test.

Table 2. Baseline and 4-weeks post-training outcomes in control and square-stepping exercise groups.

Note: presented as mean ± standard deviation. Abbreviation: baPWV, brachial-ankle pulse wave velocity; TUG, timed up and go test; 2-min, 2 minutes step test; rep, repetitions. Within group difference P-values were obtained using paired t-test. Between group difference P-values were obtained using independent t-test.

After 4 weeks of training, baPWV decreased significantly within the square-stepping exercise group (from 13.78 ± 1.97 m/s to 12.87 ± 1.55 m/s; P < 0.001), whereas it remained unchanged in the control group (13.72 ± 1.41 m/s to 13.91 ± 1.26 m/s; P = 0.093). Between-group comparisons revealed that post-intervention baPWV was significantly lower in the exercise group than in the control group (P < 0.05; Table 2). Additionally, 31% of participants in the square-stepping exercise group achieved a clinically relevant reduction in baPWV of ≥ 1 m/s.

At baseline, TUG performance was similar across the exercise and control groups, with no statistically significant differences (P > 0.05; Table 2). After the 4-week exercise intervention, the square-stepping exercise group showed a significant reduction in TUG completion time from baseline (P < 0.001). Furthermore, this reduction was significantly greater than that observed in the control group (P < 0.05).

The square-stepping exercise group exhibit a significant improvement in the 2-minute step test from pre- to post-intervention (9% increase, P < 0.001). This improvement was significantly greater than that observed in the control group (P < 0.05; Table 2).

Table 3. Correlations between changes in brachial ankle pulse wave velocity and functional variables after 4 weeks of square-stepping exercise training.

Note: Correlations were analyzed using Pearson’s correlation coefficient

The change in TUG test time was significantly and positively correlated with the change in baPWV (r = 0.61, P < 0.001; Table 3). In addition, the change in the number of steps during the 2-minute step test was significantly and negatively associated with the change in baPWV (r = -0.46, P < 0.01; Table 3).

DISCUSSION

The present study provides the evidence that a short-term, 4-week square-stepping exercise intervention elicited significant and concurrent benefits for both vascular health and physical function in middle-aged, postmenopausal women with elevated blood pressure. Notably, the alleviation in arterial stiffness was proportion to functional balance gain and aerobic capacity improvement suggest that square-stepping exercise provides multifaceted benefits for this population.

The 4-week moderate-intensity square-stepping exercise program resulted in a reduction in baPWV of 0.91 m/s, indicating a significant improvement in arterial compliance. This improvement is comparable to the ~ 0.9 m/s baPWV reduction reported by Wong et al. (2018) following 12-week of progressive stair climbing exercise and exceeds the average reduction of 0.69 m/s reported in a recent meta-analysis of exercise (Yang et al., 2024). The underlying mechanisms responsible for these vascular benefits likely involve both functional adaptations, such as enhanced nitric oxide-mediated vasodilation and reduced oxidative stress, decreased sympathetic modulation and structural adaptations including vascular remodeling (Sugiharto et al., 2023; Shing et al., 2024). Primarily, shear stress from exercise upregulates endothelial nitric oxide synthase (eNOS), promotes NO production for vasodilation, reduced vascular smooth muscle proliferation and counteraction of estrogen-deficit endothelial dysfunction (Lew et al., 2022). Concurrently, enhanced baroreflex sensitivity and vagal tone diminish sympathetic overactivity, lowering vascular stiffness (Lin et al., 2018; Sanchez-Delgado et al., 2023). Additionally, upregulated antioxidant enzymes scavenge reactive oxygen species, preserving NO bioavailability and inhibiting collagen cross-linking (Hildreth et al., 2014). These pathways synergistically improve vascular compliance. Although the baseline baPWV (~13.70 m/s) in our cohort is generally classified as within the normal range (< 14 m/s), arterial stiffness exists on a continuum (Tanaka et al., 2018). Evidence indicates that within the high-normal range, higher baPWV value are associated with greater vascular aging and cardiovascular risk. Therefore, even relatively small reduction in baPWV may represent meaningful attenuation of arterial stiffness and complications (Im et al., 2007; Yiming et al., 2017). Importantly, the magnitude of PWV reduction observed in our study approaches the 1.0 m/s threshold that is considered clinically meaningful for reducing cardiovascular risk (Vlachopoulos et al., 2012). Therefore, these findings suggest that square-stepping exercise serves as an effective strategy to counteract menopause-induced vascular impairment. The 0.9 m/s baPWV reduction should be interpreted with caution. While clinically meaningful, blood pressure fluctuations, heart rate changes, and measurement variability may partially account for this effect, complicating the attribution to exercise alone (Milan et al., 2019; Marshall et al., 2024).

In our study, the square-stepping exercise program significantly enhanced balance performance. This is an important finding, as balance commonly deteriorates in menopause due to estrogen deficiency and age-related declines in muscle strength. The effectiveness of this exercise intervention can be attributed to its biomechanical characteristics, which integrate multi-directional stepping, dynamic challenges to the base of support, and co-activation of lower-limb muscle groups, collectively improve balance control (Outayanik et al., 2018; Thanasootr et al., 2022). We also observed a significant relationship between arterial stiffness and balance ability. This finding is consistent with previous research demonstrating a positive correlation between baPWV and greater sway length in postmenopausal women (Mitsuhashi et al., 2020). Together, these results support the hypothesis that the progression of arterial stiffness contributes to balance decline. Mechanistically, increased arterial stiffness may impair microcirculation, reducing blood flow to skeletal muscles and thereby compromising balance control (Dvoretskiy et al., 2020).

The link between increased arterial stiffness and diminished exercise capacity in postmenopausal women provides a clear rationale for interventions targeting both outcomes (Figueroa et al., 2011). Accordingly, our square-stepping exercise program elicited a significant 15% increase in 2-minute step test repetitions, demonstrating an enhancement in exercise capacity. Crucially, this improvement was moderately and inversely correlated with the reduction in baPWV, reinforcing the mechanistic association between enhanced arterial compliance and physical performance. This interplay is physiologically plausible, as repeated exercise-induced hemodynamic shear stress is known to promote endothelial-dependent vasodilation and improve arterial distensibility (Green et al., 2017). Therefore, our findings present square-stepping as a dual-benefit strategy, concurrently improving physical function while addressing key markers of age-related vascular decline in postmenopausal women.

While the findings are promising, several limitations should be acknowledged. Our study is using baPWV rather than gold-standard cfPWV. However, baPWV shows strong correlation with cfPWV (r = 0.61–0.74) and comparable cardiovascular risk associations validating our baPWV results in this Asian postmenopausal cohort per Japanese guidelines (Tanaka et al., 2009; Umemura et al., 2019; Kyriakoulis et al., 2025). The 4-week intervention period, though effective for inducing short-term changes, precludes conclusions about the long-term sustainability of the observed physiological adaptations. Furthermore, our ability to elucidate the underlying mechanisms is constrained by the absence of direct measurements of variables like nitric oxide bioavailability and autonomic function. Finally, the homogenous nature of our cohort specifically middle-aged, postmenopausal women with elevated blood pressure, limits the generalizability of these results to other demographics. Future research incorporating longer interventions, comprehensive mechanistic assessments, and more diverse populations is necessary to build upon these preliminary findings.

CONCLUSION

A 4-week square-stepping exercise intervention significantly improved vascular function, balance, and functional capacity in middle-aged menopausal women with elevated blood pressure or stage-1 hypertension. Therefore, square-stepping exercise is a practical, accessible, and cost-effective non-pharmacological strategy for enhancing cardiovascular health and functional fitness in this at-risk population.

ACKNOWLEDGEMENT

We are grateful to all participants for their engagement and cooperation, which were essential for the successful completion of this study.

AUTHOR CONTRIBUTIONS

Saowanee Nakmareong: Conceptualization (Lead), Methodology (Equal), Investigation (Supporting), Writing – Review & Editing (Lead), Supervision (Lead), Project Administration (Lead), Funding Acquisition (Lead); Mattaya Thasomboon: Investigation (Lead), Formal Analysis (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Supporting), Funding Acquisition (Supporting); Eakarach Wongsaya:  Conceptualization (Supporting), Methodology (Equal), Investigation (Supporting), Validation (Lead), Data Curation (Supporting), Writing – Review & Editing (Supporting), Supervision (Supporting); Atipong Pimdee:  Conceptualization (Supporting); Methodology (Equal), Investigation (Supporting), Data Curation (Lead), Writing – Review & Editing (Supporting); Piangdaw Adchaithor: Investigation (Supporting), Formal analysis (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Supporting), Project administration (Supporting).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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