Introduction

The epidemic of cardiovascular diseases (CVDs) is currently a public health issue, which significantly contributes to global deaths and disability-adjusted life years1. Although the causes of CVDs are diverse, previous studies have reached a consensus that impaired endothelial function (EF) plays an important role2,3,4. Studies have shown that endothelial dysfunction is closely associated with the occurrence of atherosclerosis5, hypertension6,7, diabetes8,9, diabetes cardiomyopathy10,11, heart failure12,13, cardiovascular events14,15, etc. As an early marker of CVDs, EF has been used as a predictive factor for CVDs16,17 and cardiovascular events18,19. Therefore, the improvement of EF is crucial to the prevention of CVDs, and the strategies and methods for improving EF have become important concerns.

In recent decades, exercise as a means of prevention and intervention for CVDs had been included in the secondary prevention guidelines for cardiac rehabilitation20, and exercise intervention has been recognized as an important non-pharmacological strategy to promote cardiovascular health21,22. Several studies have documented the cardioprotective properties of aerobic exercise22,23,24, and the European Society of Cardiology guidelines on cardiovascular disease prevention in clinical practice have identified aerobic exercise, especially moderate-intensity continuous training, as an important approach for cardiovascular disease prevention25. The World Health Organization (WHO)26, the American Heart Association (AHA) and the American College of Sports Medicine (ACSM)27also recommend that a person should perform at least 30 min of moderate-intensity aerobic exercise per day. However, previous studies have yielded conflicting results regarding the effects of this exercise dose on EF, and limited attention has been paid to the response and adaptation process of EF to this exercise in individuals with different levels of physical fitness28,29,30,31,32,33. Flow-mediated dilation (FMD) has been the gold standard for non-invasive assessment of endothelial function34, and among these previous studies, some found that 30 min of continuous moderate-intensity aerobic exercise increased FMD in trained and untrained healthy young men28,29,30, others showed that such exercise decreased FMD in healthy young men31,32,33, and another found that such exercise did not affect FMD in healthy young adults35. Meanwhile, the effects of long-term endurance training on EF in young healthy individuals are also controversial36,37,38,39. One study found that trained endurance athletes have higher FMD than healthy young subjects36, whereas other studies found no differences in FMD between endurance trained individuals and healthy controls37,38. A recent study also found that the FMD of runners after a 2-month preparatory training period was significantly lower than that of the control group39. In terms of EF in active versus inactive individuals, one study found that athletes had larger arterial diameters but similar FMD campared to untrained individuals40; another study found that active individuals had demonstrated better vascular function and nitric oxide synthesis41; and another study observed that FMD increased in the active group and decreased in the inactive group after 45 min of exercise at different intensities (25%, 50%, 75% VO2max)42. These inconsistent results may be attributed to the age and health status of the study subjects43. There is a need for a comparative study between healthy young trained and untrained individuals, excluding the effects of individual health status and age. Furthermore, the effect should be analyzed by combining changes in basal diameter, flow-mediated maximal diameter, and blood pressure. Therefore, given these inconsistent findings, the response and adaptation of EF to a 30-min bout of moderate-intensity aerobic exercise in active and inactive healthy young men still needs to be clarified by further research.

In this study, we focused on the endurance trained college athletes and untrained college students to investigate the EF response and adaptation process to a 30-min moderate-intensity continuous aerobic exercise. Meanwhile, by comparing EF in the inactive state (before exercise) between trained and untrained individuals, we also indirectly hypothesized the effect of long-term aerobic training on EF.

Methods

Participants and design

The study was an acute intervention trial for both trained and untrained individuals. Considering the effects of age44, gender45, body size46, and health status47 on vascular function, and also taking into account that the incidence of cardiovascular disease in men is higher than that in women48, healthy young men were selected as the subjects of this study, and restrictions on body size were imposed. The inclusion criteria for the enrolment were as follows: (1) young males (18–25 years of age), (2) untrained individuals were non-physical education college students who did not participate in any form of exercise training (total exercise time < 2 h/week) and trained individuals were college athletes participating in moderate-to-high intensity endurance training (4–5 times per week, 2 h per session, total exercise training time > 8 h/week)49, (3) participants’ height should be between 170 and 185 (cm), (4) participants’ body mass index (BMI) should be 18.5–28 kg/m2 with normal blood pressure, (5) cardiopulmonary function test results should be normal without any obvious signs, symptoms, or history of chronic diseases, (6) there should be no other diseases unsuitable for physical activity, and (7) participants should not engage in smoking or drinking behavior (alcohol or coffee). Finally, 12 trained male college athletes in endurance (trained group) and 12 untrained healthy male college students (untrained group) participated in this study, and their physical characteristics are shown in Table 1.

Table 1 Participant characteristics.
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All participants had to visit the laboratory three times. In the first session, anthropometric measurements (body mass and height) and an incremental load test protocol to determine maximal oxygen uptake (VO2max) were performed after a 12-h overnight fast and 48 h of no exercise. In the second session, a 30-min run at an intensity of 60% VO2max was performed after 48 h of recovery from the incremental load test, and heart rate (HR), blood pressure (BP), and EF were measured before exercise, 30 min and 60 min after exercise. In the third session, HR, BP and EF were measured again the morning after exercise. All of these measurements were performed in a quiet, temperature-controlled room (23–25 °C) by the same professionals who were blinded to group. This study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Zhejiang Normal University (ZSRT2021015). The experimental procedure, requirements and precautions were introduced and explained in detail to the participants, and all participants agreed to participate in the research. Written informed consent was obtained from each participant before the experiment.

Anthropometric measurements

Participants arrived at the laboratory between 7:00 am and 9:00 am after a 12-h overnight fast. Upon arrival, their body weight (InBody-3.2, Biospace, Seoul, Korea) and height (YG-200, Yagami, Nagoya, Japan) were measured to the nearest 0.1 kg and 0.1 cm, respectively, in underwear and barefoot. Finally, the body mass index was calculated for each participant by dividing their weight by the square of their height (kg/m2).

Assessment of maximal oxygen uptake

The participants underwent a maximal graded exercise test on a treadmill (Pulsar model, HP Cosmos sports and medical gmbh, Nussdorf-Traunstein, Germany) to determine their maximal oxygen uptake during the week prior to the exercise experiment. After a 5-min warm-up at 6 km/h, the participants began the actual exercise protocol at 9 km/h in the trained group and 8 km/h in the untrained group. The speed was increased by 1.5 km/h every 2 min until a speed of 13.5 km/h was reached in the trained group and 12.5 km/h in the untrained group. After that, the speed remained constant while the incline increased by 1% every minute until exhaustion. Breath-by-breath expired gases were collected and analyzed with a portable metabolic analyzer K5 (COSMED, Rome, Italy). Maximum VO2 data were accepted if subjects met at least three of the following criteria: (1) being too fatigued to continue running on the treadmill, (2) having a heart rate ≥ 180 beats/min, (3) having a respiratory exchange ratio (RER) ≥ 1.10, and (4) showing either a plateau or a decrease in oxygen consumption despite an increase in load.

Assessment of blood pressure and endothelial function

Participants arrived at the laboratory at 9:00 am and sat for 15 min before performing a 30-min run at an intensity of 60% VO2max. HR, BP and EF were measured before exercise (T1), 30 min after exercise (T2), 60 min after exercise (T3), and the morning after exercise (T4). The detailed test flow is shown in Fig. 1.

Fig. 1
figure 1

Test flow for blood pressure and endothelial function before and after exercise.

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Sitting blood pressure (right arm), including systolic blood pressure (SBP) and diastolic blood pressure (DBP), was measured three times with 1-min intervals using a standard sphygmomanometer (HEM-7121, Omron Corporation, Kyoto, Japan) after 5 min of quiet rest. The mean of the two closest readings was reported as the result.

FMD, as the gold standard, is the most commonly used noninvasive clinical method to assess arterial endothelial function34. In this method, ultrasound was used to measure the diameter of the brachial arteries at baseline and after reactive hyperemia secondary to temporary occlusion, and the percentage increase in peak diameter compared to baseline was calculated. The reliability and validity of the brachial artery FMD have been confirmed as a valid marker for the assessment of cardiovascular risk50. In our study, a novel noninvasive semi-automated device (UNEX EF38G; UNEX Corporation, Nagoya, Japan) that uses B-mode ultrasound by capturing one long-axis and two short-axis images was used to determine FMD. The intra- and inter-rater reliability of the FMD procedure were acceptable51. The FMD values measured by this method showed a high correlation with the FMD values measured in the core laboratory (R = 0.868, p < 0.001), and the intra-class correlation coefficient (ICC) of the test results was also satisfactory (ICC = 0.862, p < 0.001)51. According to the protocol52, the participants in both groups remained quiet for 5 min in a supine position, and then the ultrasound probe was placed 5–10 cm above the right cubital crease, following the direction of brachial artery. After brachial artery was identified and adjusted to the clearest intima, the resting diameter (baseline) of the brachial artery was measured by computer-assessed intima-intima using the tracking gate. After completion of the resting diameter test, the occlusion cuff was inflated to 50 mmHg above systolic blood pressure and maintained for 5 min. It was then rapidly deflated to measure the maximum diameter of the brachial artery and the time to maximum diameter (TTM) within 3 min during reactive hyperemia. The images were then optimized and analyzed by the same professional operator using the UNEX EF PC analysis software (UNEX Corporation). FMD was automatically calculated by UNEX EF as follows: FMD (%) = [(maximum diameter—resting diameter)/resting diameter] × 100%.

Statistical analysis

All data were presented as mean ± standard deviation (SD). Statistical analyses were performed using IBM SPSS Statistics (Version 19.0; IBM Corp., New York, USA) and JASP Version 0.16.3 (an open-source statistical software by The JASP Team). The effects of exercise on variables of different groups were determined by mixed ANOVA. Inter- and intra-group comparisons were also performed, and effect sizes were reported for all comparisons. According to Cohen's standard, the effect size threshold is categorized as large (d = 0.80/η2 = 0.14), medium (d = 0.50/η2 = 0.06), and small (d = 0.20/η2 = 0.01)53. Statistical significance was set at p < 0.05 for all analyses.

Results

Anthropometric and physiological characteristics

The Anthropometric and physiological characteristics of the participants are shown in Table 1. Anthropometric measurements, including age, height, weight and BMI of participants, did not show significant differences between the trained and untrained groups. Maximum oxygen uptake was higher in the trained group than in the untrained group (p < 0.001).

Resting diameter

Resting diameter exhibited a large time effect (p < 0.001, η2 = 0.533; Table 2). Resting diameters at 30 and 60 min after exercise were found to be higher than those recorded before exercise in both untrained (4.05 ± 0.42 vs. 3.74 ± 0.34 mm, p < 0.01; 3.88 ± 0.38 vs. 3.74 ± 0.34 mm, p < 0. 01) and trained (4.36 ± 0.50 vs. 4.06 ± 0.38 mm, p < 0.01; 4.17 ± 0.38 vs. 4.06 ± 0.38 mm, p < 0.05) groups, but no significant difference in resting diameter was found on the morning after exercise and before exercise (Table 3).

Table 2 Effects of group and time on endothelial function, blood pressure and heart rate values.
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Table 3 Comparison of endothelial function, blood pressure and heart rate values between trained and untrained groups before and after exercise.
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Resting diameter also showed a tendency for group effect (p = 0.055, η2 = 0.157; Table 2). Resting diameters were higher in the trained group than in the untrained group before exercise (4.06 ± 0.38 vs. 3.74 ± 0.34 mm, p < 0.05) and the morning after exercise (4.09 ± 0.35 vs. 3.77 ± 0.34 mm, p < 0.05) (Table 3).

No time and group interaction effects were found for changes in resting diameter.

Maximum diameter

Maximum diameter also showed a large time effect (p < 0.001, η2 = 0.502; Table 2). Maximum diameters at 30 min after exercise were higher than before exercise in both untrained (4.34 ± 0.46 vs. 4.04 ± 0.36 mm, p < 0.01) and trained (4.70 ± 0.51 vs. 4.40 ± 0.41 mm, p < 0.01) groups, but no significant difference in maximum diameter was found on the morning after exercise and before exercise (Table 3). In fact, the maximum diameters at 60 min after exercise in the trained group were no longer different from before exercise, but those in the untrained group were still higher than before exercise (p < 0.05).

Maximum diameter also demonstrated a large group effect (p = 0.041, η2 = 0.176; Table 2). The maximum diameters were higher in the training group than in the untrained group before exercise (4.40 ± 0.41 vs. 4.04 ± 0.36 mm, p < 0.05) and the morning after exercise (4.42 ± 0.36 vs. 4.06 ± 0.36 mm, p < 0.05) (Table 3).

There were no time and group interaction effects for changes in maximum diameter.

Flow-mediated dilatation

The results demonstrated no significant effect of time on flow-mediated dilatation as evidenced by the data presented in Table 2. No statistically significant changes in FMD (mm) and FMD (%) in both the trained and untrained groups before and after exercise, as illustrated in Table 3.

A large group effect was observed for FMD (mm) (p < 0.05, η2 = 0.213; Table 2), with FMD (mm) values in the training group being higher than in the untrained group before exercise (0.34 ± 0.05 vs. 0.29 ± 0.04 mm, p < 0.05; Table 3). However, no group effect was observed for FMD (%).

Time and group interaction effects were also absent for FMD (mm) and FMD (%).

Time to maximum diameter

No statistically significant group, time, or group-time interaction effects were identified for TTM, and no notable changes in TTM were observed for the trained or untrained groups before or after exercise (Tables 2, 3).

Blood pressure and heart rate

Changes in SBP and DBP showed large time effects (p < 0.001, η2 = 0.333; p < 0.001, η2 = 0.236) (Table 2). Changes in heart rate also showed large time (p < 0.001, η2 = 0.727), group (p < 0.001, η2 = 0.687), and time-group interaction (p < 0.001, η2 = 0.304) effects (Table 2). The exercise decreased blood pressure and increased HR in both trained and untrained individuals, which gradually recovered after exercise until the morning after exercise (Table 3).

No differences between trained and untrained groups were found in blood pressure before exercise. The HR in the trained group was lower than that in the untrained group before and after exercise (p < 0.01).

Discussion

This study found that 30 min of continuous aerobic exercise at an intensity of 60% VO2max did not induce changes in FMD in either trained or untrained participants, but it did result in an increase in both resting and maximal arterial diameter that was evident 60 min after exercise and fully recovered by the next morning. These results in our study are different from those of several previous studies. Johnson et al.30 and Siasos et al.28 respectively studied 10 trained young men and 20 untrained young men, and found that a 30-min run or cycle at 50% VO2max increased FMD in both trained and untrained subjects. Similarly, a study in 18 healthy young men conducted by Tryfonos et al. found that a 30-min cycling at an intensity of 70% heart rate reserve increased not only FMD but also resting and maximal arterial diameter29. A recent study in 10 healthy young men and women also found no change in FMD after a 30-min cycling session at 60% VO2max intensity, which is consistent with our findings, but the resting diameter did not change after exercise in this study35. In addition, it is worth noting that several other studies found that a 30-min moderate-intensity aerobic exercise decreased FMD31,32,33. A study by Llewellyn et al. in 15 mixed-sex young men and women found no significant change in arterial diameter after a 30-min run at 60% VO2max intensity, but there was a decrease in FMD%31. Similarly, Caldwell et al. found the same results in eight healthy young men and women33. The study by Hashimoto et al. in nine healthy young men also demonstrated a significant decrease in FMD following a 30-min aerobic exercise at an intensity of 60% heart rate reserve, although the resting diameter was decreased32.

Our study found for the first time that resting diameter and maximal arterial diameter increased without an increase in blood pressure after a single 30-min aerobic run at an intensity of 60% VO2max, but FMD% remained unchanged. Similar to previous studies54,55, our study also showed that arterial blood pressure decreases after a single exercise session, i.e. post-exercise hypotension (PEH). PEH typically occurs in response to various types of large muscle dynamic exercise (i.e. walking, running, leg cycling, and swimming) at an intensity of 40–70% VO2max and exercise durations generally between 20 and 60 minutes54. The maximal exercise-induced reductions in systolic and diastolic arterial blood pressures have been on average 18 to 20 and 7 to 9 mm Hg, respectively, in hypertensive humans and 8 to 10 and 3 to 5 mm Hg, respectively, in normotensive humans54.

In general, the differences in the results of the existing studies were mainly manifested in whether the resting diameter and the maximal diameter increased or remained unchanged, and whether the FMD increased, remained unchanged or decreased. None of these studies found a decrease in arterial diameter after a 30-min moderate-intensity aerobic exercise. FMD is the rate of arterial dilation that is related to changes in resting and maximal diameter. We believe that the discrepancies in the results of these studies are due to statistical errors caused by inadequate sample size. This is because we observed that in certain existing studies, there was no significant difference in resting diameter and maximum diameter before and after exercise, but FMD either increased30 or decreased31,33. Indeed, the homogeneous sample sizes in these studies are small. The sample in the Johnson et al. study was 10 aerobically trained individuals, and the authors also stated that the sample size was moderate within the limitations of the study30. In the study by Llewellyn et al. the sample consisted of 8 females and 7 males31, while in the study by Caldwell et al. the sample consisted of 4 females and 4 males33. In addition, the differences in the results of these studies may also be attributed to the magnitude of changes in resting diameter and maximum diameter. If the increase in the maximum diameter is less than the increase in the resting diameter, the calculated result of FMD will decrease. For example, in the study by Hashimoto et al. the resting diameter and maximum diameter 30 min after exercise were higher than those before exercise (p < 0.05), but FMD was lower than that before exercise (p < 0.05)32. In addition, we speculate that variations in exercise mode and subject grouping may also contribute to the disparities in these findings. In these studies, the exercise mode included running and cycling, and the exercise intensity ranged from 50 to 65% VO2max, and the male and female subjects were mixed together in some cases and separated in others. It is well known that running tends to result in higher blood circulation than cycling at the same moderate intensity. Additionally, due to the effects of estrogen, women have better vascular elasticity than men, and the response and adaptation process of EF to the same exercise may differ between men and women56,57, which may also affect the results of the studies.

In fact, the “athlete’s artery” has been proposed after the “athlete's heart”58. Athletes have larger brachial artery diameters and reactivity compared to age-matched non-athletes59. Large resting brachial artery diameter has also been shown to be an independent predictor of significant coronary artery disease60. Our study also found that the resting and maximal diameters were higher in the trained individuals than in the untrained individuals, but FMD was not different between the two groups. Given that there were no differences in age, sex, height, weight, and BMI between the two groups, it can be indirectly speculated that aerobic endurance training may lead to an increase in the resting and maximal arterial diameter in young men, which implies that it may be necessary to consider changes in arterial diameter other than FMD when evaluating the benefits of exercise interventions on EF. A previous study compared male endurance athletes with untrained healthy men of the same age, height, weight, and BMI for their EF, and found that the athletes tended to have a larger resting brachial artery diameter than the untrained individuals (5.32 ± 0.37 vs. 4.79 ± 0.41 mm, p = 0.07)36. This study also found that the maximum diameter of athletes was not different from that of untrained men (6.22 ± 0.71 vs. 5.41 ± 0.59 mm, p = 0.72), but athletes had a higher FMD (%) than the latter (17.1 ± 2.3 vs. 11.2 ± 1.7%, p = 0.002)36. Such results seem to be contradictory. In general, the maximum diameter needs to increase more to achieve an increase in FMD% as the resting diameter increases. However, there was no significant difference in the maximum diameter between the trained and untrained individuals in this study. We suspect that the large individual variation in the maximum diameter after vasodilation may contribute to this result. There are also studies that found no differences in FMD between trained and untrained individuals, which is consistent with our findings37,38. Moe et al. found that the resting diameter was larger in the trained group than in the sedentary group37, but Kyte et al. found no difference in resting diameter between trained and untrained women38. Although these studies suggest that training may increase vascular diameter or FMD, it is important to note that high-intensity overtraining may still cause damage to EF. A recent study by Grandys et al. had found that after two months of preparatory training, the female athletes had significantly lower FMD compared to the female control group without training, their serum testosterone (T) and free testosterone (fT) concentrations were decreased, cortisol (C) concentrations were increased, and T/C and fT/C ratios were decreased39. Therefore, the appropriate training load based on vascular health considerations should remain a concern.

Our study found that 30 min of moderate-intensity aerobic exercise resulted in post-exercise hypotension in both trained and untrained individuals. The study also found that the heart rate of the trained group was lower than that of the untrained group before and after exercise, and the same exercise resulted in a greater increase in heart rate in untrained individuals than in trained individuals. These findings are consistent with previous studies which not only describe the response of heart rate and blood pressure to moderate-intensity aerobic exercise in individuals of different fitness levels, but also indirectly suggest the effects of exercise adaptation on heart rate and blood pressure during long-term training61,62,63.

Previous studies have also compared the differences in endothelial function and its response to resistance exercise between active and inactive individuals64,65. Morishima et al. found that an acute resistance exercise decreased endothelial function in sedentary individuals but not in strength-trained individuals with the same base brachial artery diameter and FMD64. Similarly, Varady et al. found that there was no difference in baseline FMD between sedentary individuals and weight trainers before exercise, whereas FMD was impaired in sedentary subjects but not in weight trainers after acute resistance exercise, suggesting that habitual resistance training may modulate adipokine profiles in a way that is protective against endothelial dysfunction65. It is worth noting that both studies employed high-intensity resistance exercise (75% of one repetition maximum and above), which has been shown to be less effective than low-to-moderate intensity resistance exercise in improving endothelial function66. Thus, a comparative study investigating the FMD responses to low-to-moderate intensity resistance exercise in both trained and untrained individuals remains to be performed.

Our study has several limitations. First, the study did not have a resting control group, making it difficult to determine whether the vascular changes were due to the exercise intervention itself or could have simply occurred on their own. Second, we didn’t measure shear rate and blood biomarkers, making it difficult to analyze the underlying mechanism. Finally, comparing trained and untrained individuals has the advantage of identifying a possible association between long-term training and endothelial function, but does not allow the determination of causes.

Conclusion

The response and adaptive processes of endothelial function to a 30 min of moderate-intensity aerobic exercise are consistent between trained and untrained individuals. 30 min of moderate-intensity aerobic exercise can increase resting and maximal arterial diameter in both trained and untrained healthy young men, but has no effect on FMD. In addition, pre-exercise resting and maximal diameters were higher in trained than in untrained individuals, but no difference in pre-exercise FMD (%) was found between trained and untrained individuals. This implies that long-term endurance training has the potential to increase arterial diameter, but not necessarily FMD (%), and that it may be necessary to consider changes in arterial diameter other than FMD (%) when evaluating the outcomes of exercise interventions on EF.