Effects of High-Intensity Interval Training on Improving Arterial Stiffness in Chinese Normal Weight Obese Female University Students: A Pilot Randomized Controlled Trial

Background: High intensity interval training (HIIT) has been reported to exert better effects on cardiovascular tness in obesity, but little known about the arterial stiffness (AS) in normal weight obesity (NWO) females. Thus, this study investigated the changes of body composition, heart rate (HR), blood pressure (BP), blood lipids as well as the parameters of propensity for AS (arterial velocity pulse index (AVI) and arterial pressure volume index (API)). Methods: Forty NWO female university students were randomly assigned to the control group (n=20) or the HIIT group (30 minutes at 90% HR max , n=20). The participants in HIIT group accepted a four-week HIIT intervention for ve days a week. The above parameters of body composition, cardiometabolic biomarkers, lipid prole, and AS were assessed before and after the HIIT intervention. Results: HIIT signicantly improved the body composition in decreasing the BMI (p=0.001), BF% (p<0.001), BFM (p<0.001), LBFM (p<0.001), LMC (p=0.016), and Obesity degree (p=0.001), and increased the SMM (p=0.020), Protein content (p=0.035), TBW (p=0.017), FFM (p=0.019), BCM (p=0.020), and Inbody score (p<0.001). HIIT also affected the cardiometabolic biomarkers by decreasing the HR (p=0.001), SBP (p<0.001), and DBP (p<0.001). As for the lipid prole, HIIT obviously ameliorated the blood lipids metabolism by decreasing the levels of TC (p=0.001), TG (p<0.001), LDL (p<0.001), and TC/HDL (p<0.001), and increasing the levels of HDL (p<0.001). In addition, the AS was improved by HIIT intervention through decreasing the AVI (p<0.001) and API (p<0.001). Conclusions: Our results provided herein demonstrate that this pattern of a 4-week HIIT had a positive impact on physical tness in the aspect of body composition, lipid prole, and cardiovascular function in NWO female university students.

been performed mainly in Japan, there have been no reports about using them to evaluate the AS of NWO female university students in China.
Although increasing evidence suggests that exercise is the most predominant non-pharmacological strategy for attenuating excessive VAT deposition and ght obesity and related complications [19], NWO female university students prefer diet to exercise. Among the main barriers for inactive individuals to physical activity and maintain a physically active lifestyle are the reported lack of time, access to sporting facilities, the nancial costs of exercising, and the inability to adhere to lengthy exercise programs. Recently, as an emerging and promising, highly e cient, multi-stimulated, cost-saving means, and timeeffective pattern of exercise intervention, high-intensity interval training (HIIT) is born at the right moment. HIIT involves several short sets of high-intensity bursts (≥ 80% of HRmax or ≥ 90% of maximal oxygen uptake, often reaching anaerobic threshold) interspersed with low-intensity recovery or rest (65-80% of HRmax) [20,21]. A growing body of evidence demonstrated that HIIT can be an effective alternative to traditional moderate-intensity continuous training (MICT). Compared with MICT, HIIT has unique advantages in treating overweight/obesity people, because it not only could lead to greater adipose tissue loss, more effectively reduce abdominal and visceral fat mass, and improve the body composition and dyslipidemia [22][23][24], but also could mediate larger decreases in systolic blood pressure [25], improve cardiorespiratory and vascular function, and reduce risk factors of CVD [22,26]. Moreover, it's convenient and enjoyable for the public since the sport events and modes of HIIT are various and not limited to the speci c stadium [27,28]. As a result, HIIT has high unsupervised adherence rates in overweight and obese adults. However, despite clear evidence for the positive vascular function and cardiovascular adaptations following HIIT, it's still unclear whether HIIT could yield a bene cial effect on AVI/API and AS by improving the body composition and lipids metabolism of NWO female university students.
NWO female university students are a special population. In this study, we selected them as the research object, mainly aimed to evaluate the possible changes of their body composition and cardiovascular function, and then investigate and analyze the potential clinical effectiveness of short time HIIT in the treatment of the above changes.

Study participants
We enrolled one hundred and thirty-seven female college students with normal BMI (18.5-24.9 kg/m 2 ) at the Shanghai University of Medicine and Health Sciences (Shanghai, China) from November to December 2020 after the exercise risk assessment. The experiment protocol was submitted to and approved by the institutional review board of the Shanghai University of Medicine and Health Sciences (2020-20YJCZH001-03), and all participants gave written informed consent before the study after procedures, associated risks, and potential bene ts of participation had been explained, then the study was performed in accordance with the principles outlined in the Declaration of Helsinki.
After the exercise risk assessment, forty NWO subjects were screened using body composition measurement apparatus (InBody-770, Biospace Co., Seoul, Korea) according to the criteria of female NWO (BF%>30%, which is the NWO obesity cut-off for Asian adults) [10]. Participants were randomly divided into Control group (n=20) and HIIT group (n=20). Subjects in the control group did not take regular exercise and only participated in physical education once a week, while subjects in the HIIT group performed duration of 4-week HIIT. Both two groups were not interfered with the daily diet and other daily living behaviors. Outside implemented program, all participants were instructed to maintain their normal levels of physical activity and normal dietary patterns (and not to use any dietary supplements). 10 of the 40 participants (7 in the control group and 3 in the HIIT group) resigned from the study during the intervention due to personal reasons. Flow-chart diagram is detailed in Figure 1.

HIIT Intervention protocol
Participants in the HIIT group were familiarized with a week acclimatization training to acquaint the training session, learn the motor movement, and gradually adapt and increase the training intensity. After familiarization, the participants completed 4-week. The frequency of HIIT was ve times a week. The HIIT intervention began with 10 min of warm-up (including the stretch of self-propelled lower extremity muscles, the world greatest stretch, and the rotator cuff muscle training) to prevent the sports injury. Then the training was performed with 9 min of high-intensity exercise combined motion (jumping jacks, runups, squat jump, burpee, high elbow plank, step jump, double knee lifts, mountain climbers, lunges in place; 40 s for each movement interspersed with 20 s for interval rest) followed by 1 min of recovery rest and this was repeated 3 bouts and ended with 10 min static stretching of upper and lower limb muscles. The intensity of training corresponded to 90% of the maximum heart rate (HR max ) which was monitored by Prince-100H (Heal Force, Shanghai, China) during the entire training session. The entire HIIT process is guided by professional coaches and supervised by athletic instructor in case of sports injury or exercise fatigue. HIIT intervention protocol is detailed in Figure 2.

Body composition
Subjects were given complete instructions on the body composition analysis procedure and were instructed not to make any intense physical effort in the 8 h prior to body composition measurement. Body composition (Body weight; BMI; BF%, body fat percent; BFM, total body fat mass; LBFM, BFM of left arm; SMM, total body skeletal muscle mass; Protein; BMC, bone mineral content; FFM, fat free mass; LFFM, FFM of left arm; TBW, total body water; LTBW, TBW of left arm; LMC, Measured circumference of left arm; BCM, body cell mas; Inbody score, and Obesity Degree) was detected by InBody-770 (Cerritos, CA, USA) multi-frequency bioelectrical impedance (BIA) device before and after the 4-week HIIT intervention. The subjects held the handle with both hands and abducted the upper limb 30° during the measurement. They were asked to take off their shoes, socks, hats, and heavy coats. Electronic products (e.g., mobile phones, earphones) and nger ring were also not permitted to carry. Handles and metal pedals were wiped with an alcohol cotton ball before each subject was tested.

Blood lipids
Fingertip blood samples (35 μL for each subject) were collected by capillary tube. Then they were added to a blood lipid test card quickly and examined by Cholesterol Monitoring system CCM-111 (Acon Biotech, Hangzhou, China) to detect the concentration of lipid pro les (TC, total cholesterol; TG, triglyceride; HDL; LDL; and TC/HDL).
Resting heart rate, blood pressure, and arterial stiffness The left arm of resting heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), AVI, and API were all simultaneously measured using the PASESA AVE-2000 PLUS (Shisei Datum, Tokyo, Japan) with the subject in the sitting position. Measurements were taken in a quiet room with the temperature maintained at 20-26 ℃. The above parameters were calculated before the intervention and the 7:00 am the day after HIIT completion. The subjects were forbidden drinking coffee and kept calm and stable during the measurement for avoiding the uctuation of blood pressure.

Statistical analysis
All data are expressed as mean ± standard error (SE). The repeated measures ANOVA (group main effect and time by group interaction) within-between interaction design was used to evaluate indexes changes pre to post intervention through SPSS 26.0 software. If the interaction between time and group is signi cant, simple effect test analysis is needed. An alpha value of p<0.05 was accepted as the minimal level of signi cance.
Before and after the intervention, the within-group comparisons showed that the HIIT group had signi cantly decreased Body weight (p=0. Effects of HIIT on heart rate and blood pressure of NWO females As shown in Table 2, we found a signi cant time×group interaction in the aspects of resting HR (F (1,28) =37.780, p<0.001, η 2 =0.574), SBP (F (1,28) =23.125, p<0.001, η 2 =0.452), and DBP (F (1,28) =41.165, p<0.001, η 2 =0.595). There was no signi cant difference in HR, SBP, and DBP between the two groups before the HIIT intervention. However, compared with the control subjects after the HIIT intervention, the HR (p=0.001), SBP (p<0.001), and DBP (p<0.001) were obviously decreased in the HIIT group. Before and after the intervention, the within-group comparisons showed that the HIIT group had signi cantly decreased HR (p<0.001), SBP (p=0.029), and DBP (p=0.006), while the Control group had exactly opposite signi cant changes.

Discussion
There is strong evidence linking NWO and increased CVD and all-cause mortality, and women with NWO were 2.2 times more likely to die from CVD compared with those with low BF [10]. Female university students are the high incidence but underdiagnosed and understudied group of NWO. In this study, we used the bioelectrical impedance analyzer InBody-770 to measure and estimate the body composition and found, if left untreated, some signi cantly progressive adverse changes in NWO female university students, such as increased Body weight, BMI, BF%, BFM, BFM of left arm, and obesity degree, which will may aggravate the potential cardiovascular health hazard. Early numerous studies have validated that elevated BF% and BFM are associated with disturbances in lipids metabolism and cardiometabolic [29], which could impair cardiovascular function. Herein, if left untreated, we also found NWO females had obviously lifted resting HR, SBP, DBP, TG, and LDL over time. The normal range of SBP and DBP is respectively less than 120 and 80, however, both SBP and DBP were excessive in NWO females, implying the risk of hypertension. There was a study pointed out that the NWO subjects demonstrated left ventricle systolic and diastolic dysfunction, increased brosis intensity [30], so we recommend both systolic and diastolic BP should be evaluated to better de ne the metabolic and vascular pro le of NWO females.
Previous evidence has shown that higher BMI, BP, TG, and LDL may cause persistent endothelial damage, which will lead to endothelial dysfunction and AS increase, and this is considered the rst step in the progression of atherosclerosis [31]. Elevated HR is an important determinant of the mechanical properties of the vascular atherosclerotic lesions, which is related to the development and progression of vasculopathy [32,33]. Two key points of HR elevation contribute to atherosclerosis: rstly, the elevated HR increases the oscillatory shear stress to lower the arterial distensibility. Secondly, it intensi es the pulsatile motion of the heart to hinder the local hemodynamics [34]. BP has been reported to be mediated by AS [35]. For example, elevated SBP is signi cantly associated with higher central arterial stiffness in healthy, normotensive men and women[36].
Blood-based biomarkers such as TC, TG, LDL, and HDL are well-established indicators of cardiovascular function risk. In this study, before the intervention, we found that the values of TC (suitable range < 5.20 mmol/L, edge elevated 5.20-6.20 mmol/L), TG (suitable range < 1.70 mmol/L, edge elevated 1.70-2.30 mmol/L), and LDL (suitable range 2.60-3.40 mmol/L, edge elevated 3.40-4.10 mmol/L) were within the edge elevated range, and HDL (suitable range ≥ 1.00 mmol/L, abnormal range < 1.00 mmol/L) were within the abnormal range in NWO females. Similarly, if left untreated, these indicators were further elevated or were abnormal. The characteristic of atherosclerosis is visible atherosclerotic lesions in the artery walls, especially in the coronary artery and aorta. These lesions primarily occur due to excessive lipid deposition [37]. Therefore, atherosclerosis originates from the passive diffusion of circulating LDL through endothelial junctions into the vessel intima [38]. One of the most representative is the elevated levels of TC and LDL [37]. The reduction of LDL can further lower cardiovascular mortality. TG is the major component of triglyceride-rich lipoproteins (TRLs), which needs to combine with associated proteins into lipoprotein particles [39]. Recently, some studies report that TG and TC content within TRLs may contribute to the development of atherosclerotic cardiovascular disease [40,41]. In addition, HDL enhancing foam cell cholesterol e ux is considered as the rst step of reversing TC transport, which is a promising antiatherogenic strategy [42]. The sensitivity of TC/HDL value is higher than the pure TC or HDL, which is used to independently predict the severity of fatty liver caused by obesity, atherosclerotic, and coronary heart disease [43].
AS is considered one of the earliest detectable measures of vascular damage. However, it's very di cult to directly measure AS. In the last decade, a variety of non-invasive methods and devices such as CAVI, baPWV, cfPWV, IMT, and aortic augmentation index (aAI) has been used to evaluate atherosclerosis by assessing AS or impaired endothelial function. But currently available devices and methods are unsuitable for the patient screening or risk strati cation in routine clinical practice owing to factors such as the postural requirements, variability among investigators, and time-consuming manual procedures. Recently, AVI and API, two novel non-invasive vascular indexes for evaluating systemic and peripheral AS, respectively, have been gaining attention. They are convenient, quick (about 1 min), and comfortable for systemic endothelial function and AS assessment using cuff oscillometry technologies and suprasystolic cuff oscillometric wave measurement (PASESA AVE-2000 PLUS, Japan) in the clinical setting. The AVI mainly re ects the AS from the aorta to the muscular artery and peripheral resistance, which is signi cantly associated with BMI, abdominal circumference, and TG [44]. While API mainly re ects the stiffness of the brachial artery, which is affected by coronary stenosis, arterial compliance and more sensitive to arterial SBP [45]. Meanwhile, they are both not only associated with baPWV, preclinical carotid arterial compliance, as well as AS in general populations [16,44], but also with the number of cardiovascular risk factors, the Framingham risk score[46], and CAD [47]. These two new indexes are therefore expected to be useful clinically for predicting and evaluating the future risk of atherosclerotic diseases and cardiovascular events. However, data on subjects with NWO females remain scant, not even available in China.
AVI and API have emerged as surrogate markers of cardiovascular disease in obesity and a predictor for CVD events in adulthood. According to the manufacturer's instructions, the normal range of AVI is 11 to 15 (age 18-23), and the API is 15 to 22 (age [15][16][17][18][19][20][21][22]. In the current study, we found both AVI and API exceeded the normal range in NWO female university students. Even worse, the API further increased in the Control group due to they did not get effective intervention. Combined with the abnormal body composition, BP, and lipids metabolism, these results strongly suggested that the NWO females have an increased risk of arteriosclerosis and CVD. Early detection of the degree of arteriosclerosis in the NWO population and timely intervention are of great signi cance for preventing and treating cardiovascular and cerebrovascular diseases. The previous study has revealed that the enhanced AVI represents increased workload on the heart with elevated central BP and is highly correlated with the augmentation index. API is a useful predictor of future cardiovascular disease, which is independently associated with both the Framingham Cardiovascular Risk Score and the Suita Score[46]. Fujiwara et al. found that AVI and API were higher in patients with coronary artery disease and positively correlated with the severity of coronary artery stenosis [14]. Therefore, the higher values of AVI and API represent the higher propensity for AS and assess the potential risk of CVD in the NWO population. The mechanisms of NOW increasing AS and impairing the vascular function ascribe to chronic high fat accumulation, which stimulates multiple pro-in ammatory cytokines and elevates NOX-mediated ROS production, induces oxidative stress, provokes vascular endothelial cells functional disorder, and disturbs the secretion of vascularizing factors (nitric oxide synthase and nitric oxide) and vasoconstrictor factor (Ang-and endothelin, vasoconstrictor) [48,49]. Moreover, perivascular adipose tissue also seems to promote local in ammation and reduce capillary density and microvascular nitric oxide (NO, vasodilator) bioavailability to resulting in impaired endothelial function and vasodilatation, which will eventually accelerate the atherosclerotic process at an early stage [50].
Prescribing physical activity to people with obesity is no longer a rarity and in fact has become recognized as a necessity. In recent years,HIIT is among the most highly recommended measures for obesity because it is well tolerated and favorably affects cardiometabolic risk factors. Our study showed that 4-week HIIT effectively reduced the Body weight, BMI, BF%, BFM, LBFM, and obesity degree while increased SMM, protein content, BMC, FFM, LFFM, TBW, LTBW, BCM, and Inbody score in the HIIT group, only LMC had no difference. Interestingly, after HIIT intervention, most of the results of the between-group comparisons were in line with the within-group of the HIIT group except for no difference of body weight, BMC, LFFM, and LTBW. The main reason may be possibly due to the difference in sample size between the two groups (HIIT n = 17 vs. Control n = 13), it's also possible that the statistic method of repeated measures ANOVA may be overly strict. Nevertheless, based on these results, we were still able to demonstrate that short-term HIIT could be a potent stimulus for effectively improving the body composition in NWO females. Previous evidence allows us to speculate that these positive improvements in body composition after HIIT training were likely the result of an upregulation of bioenergetic oxidation (especially fat oxidation) and energy expenditure due to excess postexercise oxygen consumption (EPOC) [51,52]. HIIT consumes large amounts of glycogen during exercise, so more fat will be oxidized during the recovery period to resynthesize glycogen. And, HIIT training may mobilize quite a bit of skeletal muscles, which also may activate the skeletal muscles' catabolism signaling mechanisms (especially lipodieresis) and increase muscle protein synthesis [53,54]. Besides these, HIIT could promote the secretion of catecholamines, epinephrine, norepinephrine, and growth hormone, which will accelerate fat decomposition to achieve effective fat and body weight loss [26].
Previous studies elucidated that HIIT could decrease BMI, BF, and BFM in overweight and obesity [22,55], and HIIT elicited superior bene ts than MICT in weight control, fat loss (especially abdominal and visceral fat), FFM, and SMM in both healthy and chronic illness populations [56][57][58]. Excessive amounts of cardiac adipose tissue are reported to be associated with CVD. HIIT has been shown to even reduce epicardial adipose tissue mass and cardiovascular risk in physically inactive participants with abdominal obesity [59]. The results imply that HIIT is a sustainable and training strategy for improving weight management. Furthermore, although generating a similar magnitude of improving BFM and waist circumference modest as the MICT does in overweight and obese individuals, HIIT can save nearly 40% time commitment each week[60]. Therefore, HIIT is considered as a time-e cient and effective exercise strategy for managing overweight and obesity.
However, to the best of our knowledge, little is known regarding the effects of HIIT on CVD factors, namely BP, lipids metabolism, and AVI/API, in NWO female university students. Firstly, BP is closely correlated with cardiovascular health, and hypertension has led to high cardiovascular morbidity and mortality worldwide [61]. Some studies have shown that HIIT had a superior function of decreasing resting HR, SBP, and DBP[62-64], and was associated with greater improvements in dealing with hypertension when compared to MICT among the hypertensive patients and the overweight/obesity adolescent girls[65-67]. These results are concordant with our ndings. Secondly, HIIT has been reported to have the function of redressing lipid metabolism disorders in obesity[68, 69] and may be a preferable therapy for atherosclerosis. Our results demonstrated that 4-week HIIT could signi cantly reduce TC, TG, LDL, and TC/HDL, and raise HDL in the HIIT group, and the changes of these indexes were consistent with that of when compared with the controls. More importantly, HIIT could adjust these indexes to the normal ranges, and our nding was consistent with Fisher et al in overweight and obese young men [70]. Gripp et al. reported that most of the positive effects of the HIIT were also found to be longer-lasting and maintained after the suspension for 4 weeks [71]. Accumulation of LDL inside the blood vessels serves as a major cause of arteriosclerosis, while HIIT can prevent LDL accumulation [26]. Moreover, HIIT also can affect HDL function, including the promotion of reverse cholesterol transport and lipid peroxide transport clearing [72]. Therefore, this pattern of short-term HIIT will bene t the NWO females from ameliorating lipid metabolism.
Thirdly, although the effects of aerobic exercise on reducing AS and CVD risk have been investigated before, the relationship between AVI/API and HIIT improved NOW females AS has not been studied fully.
Here, we examined for the rst time the effect of HIIT on the two new arterial indices on AS in Chinese NWO female university students. We veri ed that HIIT could signi cantly lower the values of AVI and API to the normal ranges, and the fully automatic and rapid measurement of AVI/API may enhance the practical value in a large population to identify subjects at high risk of atherosclerosis. AS is mainly in uenced by vascular endothelial function, HIIT has well-established bene cial effects on endothelial function in overweight men/adolescents and obese young women [73,74]. Adaptive changes in endothelial function have been shown to depending (to a large extent) on the activity of eNOS, which induces NO production to relax smooth muscle cells, capillaries, and small arterioles, subsequently dilates blood vessels and increases arterial compliance in the muscular arteries [75,76]. HIIT has been reported to increase endothelial eNOS protein content and NO availability, and caused signi cant improvements in brachial artery endothelial-dependent dilatation and aortic stiffness in obese individuals with elevated CVD risk [77]. Additionally, a meta-analysis showed that HIIT was associated with up to a twofold increase in endothelial dilator function at the macrocirculatory level when compared with MICT in adults with metabolic and cardiovascular disease [78].
However, the mechanism of reducing the propensity for AS is still unclear in our study. There are many gaps in the literature on NWO unlike for overt obesity and further study should disclose the underlying relationship between the HIIT and the vascular adaptation in improving the AS in NWO female university students and explore the molecular mechanisms. It should be noted that the exercise risk assessment needs to be carried out before the formulation of HIIT prescription, and the adaptive training along with the increasing intensity and su cient warm-up, relaxation, and cooling-down are essential for avoiding sports fatigue and sports injuries even the concurrently health risk. In this study, we did not observe any adverse exercise events (e.g., muscle injury, overfatigue, syncope, palpitation, angina, abnormal uctuation of blood pressure, nausea, vomiting, dyspnea) during the HIIT intervention. Hence, HIIT utilization is safe for NWO females. Last but not least, large, multicenter, and prospective studies are required to establish the optimal HIIT protocols for NWO female university students.

Limitations
The study was designed as a prospective, randomized controlled trial (RCT) exercise intervention trial comparing the effects of a 4-week HIIT training program on NOW female university students. While, this study has some limitations that should be considered before interpreting the results. First, the sample size was relatively small and the control group had a higher attrition rate (35%), probably because they didn't see the weight loss or bene t. In contrast, the HIIT group had high adherence. Even so, it did not prevent us from seeing clear differences between the two groups, and the magnitude of the bene cial effects of HIIT on NWO females' AS was adequate. Second, although it has been reported that HIIT may result in suppressed appetite[79], participants were instructed to not alter their diet behaviors for the duration of the intervention, change in habitual energy intake was not monitored, which may affect the nal results. Third, we did not examine the arterial compliance, re ected wave, vascular endothelial function, or female hormones, which could have important effects on AS.

Conclusion
All in all, our observations suggest that HIIT is an effective and acceptable activity for NWO female university students, and we provide up-to-date evidence on the impact of short-term HIIT in obviously improving body composition and abnormal lipid metabolism, reducing BP and AVI/API, as expected.
These changes caused by HIIT will be hugely bene cial for reducing AS and CVD risk, and enhancing NWO female university students' physical tness and well-being. Tables Table 1 Effects Table 4 Effects of HIIT on AVI and API of NWO females Data are presented as mean±standard error (SE). ** p<0.05, HIIT group (post) vs. Control group (post); ## p<0.01, pre vs. post in HIIT group; △ p<0.05, pre vs. post in Control group. AVI, Arterial velocity pulse index; API, Arterial pressure volume index. Figure 1 Flow chart diagram. The recruiting and screening process of normal weight obesity (NWO) female university students for study protocol. The assessment indicators include the body composition, lipid pro le, and cardiovascular function.