Effects of an Aerobic Exercise Intervention Program on Arterial Stiffness and Inflammation in Women With Systemic Lupus Erythematosus
Phase: NA
Conditions: Systemic Lupus Erythematosus
Sponsor: Fundación Pública Andaluza para la Investigación Biomédica Andalucía Oriental

Effects of 12-week Aerobic Exercise on Arterial Stiffness, 
Inflammation, and Cardiorespiratory Fitness in Women with Systemic 
LUPUS Erythematosus: Non-Randomized Controlled Trial 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NCT: NCT03107442 
 
 
 
Document Date: 26/03/2024 

Study Protocol and Statistical Analysis Plan 
1. Objective 
The primary aim of this study was to assess the effect of a 12-week aerobic exercise 
intervention following the ACSM guidelines on arterial stiffness in women with SLE in 
comparison with usual care. Secondary aims were to assess the effects of the exercise 
intervention on inflammation, oxidative stress, and cardiorespiratory fitness. 
2. Design and Protocol Registration 
This non-randomized controlled trial was registered at clinicaltrials.gov [NCT03107442] 
on 11 April 2017, before the enrolment of participants started (i.e., on 12 April), and no 
deviations occurred regarding the primary outcome and the secondary outcomes analyzed 
here. 
3. Setting and Eligibility Criteria 
Participants were recruited from the Systemic Autoimmune Diseases Unit of the “Virgen 
de las Nieves” and the “San Cecilio” University Hospitals. Women with a diagnosis of 
SLE according to the ACR criteria [26], a follow-up of  12 months, clinical and treatment 
stability during the previous six months, and not performing regular exercise (defined as 
 60 min/week of structured exercise) were included. Exclusion criteria were to have been 
under biological treatment in the previous six months or to need a prednisone dose of >10 
mg/day; a background of CVD in the previous year; to present contraindications to perform 
exercise; other associated rheumatic conditions; pregnancy; active acute or chronic 
infection; neoplasms; acute renal failure; cardiac or pulmonary involvement; body mass 
index (BMI) >35; or not being able to read, understand, and sign written informed consent. 
All participants received detailed information about the study procedures, and signed 
written informed consent. The Research Ethics Committee of Granada approved the 
protocol on 11 November 2016 (reference No.: 10/2016). 

4. Procedures 
A telephone screening was conducted. Potentially eligible participants were invited to a 
personal screening and, if included, day 1 of the baseline examination was performed. The 
baseline examination comprised two assessment days. On day 1, pulse wave velocity 
(PWV) was assessed. Thereafter, cardiorespiratory fitness testing was performed, and 
socio-demographic and clinical information was collected. On day 2 (i.e., between two and 
four days after day 1), 8-h fasting blood samples were collected between 8:00 a.m. and 
10:00 a.m. This article follows the TREND statement for improving the reporting of non-
randomized experiments of behavioral and public health interventions (downloadable at 
EQUATOR Network: https://goo.gl/ZSyLrj; Supplementary Table S1) [27]. The funding 
source had no role in the study. 
5. Methods 
5.1.Interventions 
5.1. 1. Exercise Group 
To maximize transparency and replicability, the exercise program described in this 
manuscript follows the Consensus on Exercise Reporting Template (CERT; 
Supplementary Table S2) [28]. The patients assigned to exercise performed two 75-min 
sessions per week during a total of 12 weeks (i.e., 24 sessions) of moderate to vigorous 
intensity aerobic exercise on a treadmill (BH, Serie i.RC12 Dual, Vitoria-Gasteiz, Spain) 
from 24 April to 14 July 2017. The sessions took place in a quiet room of the “Virgen de 
las Nieves” Hospital, Granada (Spain). 
All sessions were performed in groups of a maximum of five persons (depending on the 
patients’ schedule preferences) and were supervised by both exercise professionals with a 
degree in Sports Sciences and residents from the Internal Medicine Department.  
 
 

Attendance at the sessions was registered daily and patients were contacted upon any 
missing session to ask for the reason and motivate them to replace it on an alternative day 
of the same week. Adherence to exercise is reported as the median attendance frequency 
and the proportion of patients attending >75% (i.e., 18 sessions; the minimum pre-defined 
attendance to assess efficacy) and >90% of the sessions. All the sessions began with a warm-
up comprising 3–4 min of activation on the treadmill at about 35–40% of the heart 
rate reserve (HRR) and 3–4 min of active stretching of major muscle groups, and ended 
with a cool down phase of static stretching of major muscle groups and relaxation. Exercise 
was individually prescribed to represent moderate-to-vigorous intensity, with training 
intensity ranging from 40% to 75% of each patient’s HRR. The maximum heart rate 
(HRmax) was estimated with the formula by Tanaka et al. (HRmax = 208 x (0.7 x age)) 
[29]. The training (or target) heart rate (tHR) was calculated with the formula tHR = HRrest 
+ (%HRR). Heart rate was continuously monitored during all sessions (Polar V800, 
Kempele, Finland). We used the session rating of perceived exertion (RPE) as a measure of 
subjective training load [30], and the feeling scale to assess positive affective responses 
experienced before and after each session [31]. 
The starting level was specific for each individual according to her previous exercise 
experience and physical fitness. During the first half of the program, only continuous 
exercise was performed so that the patients got used to the treadmill and felt confident at 
increasing intensities. Continuous sessions comprised several bouts of exertion at constant 
intensity, followed by a couple of minutes of recovery (i.e., rest) to drink water. During 
month 2, there were alternated continuous and interval sessions, and at month 3, the patients 
undertook interval training sessions, where there were periods of lower and periods of 
higher intensity efforts followed by some minutes of rest for hydration (Table 1). The 
progression in volume and/or intensity was patient-limited and was undertaken by 
increasing the treadmill speed (first) or inclination according to the symptoms and perceived 
exertion. There were no home-based or non-exercise components within this intervention. 
However, if a participant was eventually not able to attend a particular session, we provided 

her with a heart rate monitor and allowed recovery of that session out of the Hospital (a total 
of seven sessions were recovered in this fashion). Finally, the exercise intensity progressions 
had to be slightly modified from the initial plan. For instance, several patients perceived a 
5% HRR intensity increase (i.e., from one week to another) as very heavy and difficult-to-
follow. Consequently, there were weeks in which exercise intensity increased by 2.5% 
instead of 5% (Table 1). 
5.1.2. Control Group 
After the baseline evaluation, the SLE patients assigned to the (usual care) control group 
received verbal information about a healthy lifestyle, including physical activity guidelines 
and basic nutritional information. 
 
5.2. Outcome Measures 
Primary Outcome Measure: Arterial Stiffness 
Arterial stiffness was assessed in a sitting position by PWV [9], using the Mobil-O-Graph® 
24 h pulse wave analysis monitor (IEM GmbH, Stolberg, Germany), whose operation is 
based on oscillometry recorded by a blood pressure cuff placed on the brachial artery. The 
coefficient of variation (CV) of Mobil-O-Graph for consecutive PWV analyses is 3.4% and 
its intraclass correlation coefficient is 0.98 [0.96–0.99] [32]. This device has been largely 
shown to be valid and reliable for measuring PWV and central blood pressure in different 
populations [33,34], meets the accuracy requirements of the 
British Hypertension Society (BHS) standard [35], and can be recommended for clinical 
use [36]. 
 
 
 
 
 
 

Secondary Outcome Measures 
Blood Samples and Biochemical Analyses 
Fasting blood specimens for biochemical and immunological tests were collected and 
routinely processed by the central laboratory of our hospital. Among other measurements, 
they included lipids, insulin (BioRad, Marne-la-Coquette, France), and a routine 
biochemical profile. The homeostatic model assessment for insulin resistance (HOMA-IR) 
was calculated (HOMA-IR = glucose (mmol/L)   insulin ( U/L)/22.5). 
Inflammatory 
Markers 
Serum 
high-sensitivity 
CRP 
was 
assessed 
by 
an 
immunoturbidimetric method using the ARCHITECT cSystems (MULTIGENT CRP 
Vario assay); the limit of quantitation was 0.2 mg/L and the upper limit for normal serum 
was 
5 
mg/L 
(coefficient 
of 
variation 
<6%). 
Interleukin 
6 
and 
TNF- 
, as well as myeloperoxidase (MPO; as marker of oxidative stress), were measured in 
plasma. Serum was initially separated by centrifugation and stored at 􀀀70 C.  
Bioserum 
concentrations 
of 
IL-6/TNF- 
 (pg/mL) and MPO (ng/mL) were measured by an immunoradiometric assay using 
commercial kits (MILLIPLEX MAP Kit Human High Sensitivity T Cell Magnetic Bead 
Panel (HSTMAG-28SK) and Human Cardiovascular Disease Magnetic Bead Panel 2 
(HCVD2MAG-67K)), Millipore) following the manufacturer’s instructions. Quantitative 
data were obtained by using the Luminex-200 system (Luminex Corporation, Austin, TX, 
USA), and data analysis was performed on XPonent 3.1 software (Austin, TX, USA). The 
detections limits were 0.73 pg/mL for IL-6, 0.43 pg/mL for TNF-a, and 0.024 ng/mL for 
MPO. 
Cardiorespiratory Fitness 
Cardiorespiratory fitness was assessed with the Bruce submaximal treadmill protocol [37]. 
The test comprised five increasing workload stages of 3 min each (stage 1: 2.7 km/h and 
10% inclination; stage 2: 4 km/h and 12% inclination; stage 3: 5.5 km/h and 14% 
inclination; stage 4: 6.8 km/h and 16% inclination; stage 5: 8 km/h and 18% inclination). 
The test concluded when the participant achieved 85% of the individual’s HRmax, as 

estimated with the formula by Tanaka et al. [29]. As validated SLE-specific formulas to 
estimate VO2max are not available, we used the total time to reach 85% HRmax as the 
outcome of interest. 
 
Other Measurements 
All participants filled out a socio-demographic and clinical data questionnaire. Height (cm) 
was measured using a height gauge, weight (kg) with a bioimpedance device (InBody R20, 
Biospace, Seoul, Korea), and body mass index (BMI) was calculated (kg/m2). Blood 
pressure was measured with Mobil-O-Graph® (IEM GmbH, Stolberg, Germany) [36]. 
Disease activity was assessed through the Systemic Lupus Erythematosus Disease Activity 
Index (SLEDAI, range 0–105 where a higher score indicates higher degree of disease 
activity). Physical activity was self-reported at baseline and at week 12 with the 
International Physical Activity Questionnaire [38]. 
 
Sample Size 
The sample size was calculated for the primary outcome (i.e., PWV). Ashor et al. found an 
average effect of aerobic exercise on PWV of 􀀀0.63 m/s in adults aged  18 years [21]. A 
total of 52 patients (26 per group) were needed to detect an effect of 􀀀0.63 (SD 0.75) m/s, 
with a power of 85% and an error of 0.05. Anticipating a maximum loss to follow-up of 
15%, we aimed at recruiting a total of 60 patients. 
 
Treatment Allocation and Blinding 
Randomization was not feasible because more than half of the patients who regularly attend 
the Autoimmune Disease Units lived far from the Hospital and were not able to attend 
twice per week in case of being randomized to exercise. Therefore, participants from the 
city of Granada were included in the exercise group and participants living outside Granada 
were included in the control group. 
 

To minimize potential selection bias, we aimed to match the groups by age ( 2 years), 
BMI ( 1 kg/m2), and SLEDAI ( 1 unit). The data analyzer was blinded to the patient 
allocation. 
 
Statistical Analysis 
The distribution of the main study variables was assessed through histogram and Q-Q 
plots. As the main outcomes were non-normally 
distributed, their descriptive 
characteristics were presented using the median and interquartile range instead of the 
mean and standard deviation and we used non-parametric tests for the main analyses. 
Between-group baseline characteristics were compared with the Student t-test (when 
normally distributed) or Kruskal-Wallis test (when non-normally distributed) for 
continuous variables and the Chi-square test for categorical variables. The between-group 
differences in the studied outcomes were assessed through quantile regression with 
baseline values, resting heart rate (bpm) [39], and changes in physical activity (min/week; 
since the change in self-reported physical activity at week 12 was >60 min higher in the 
control compared to the exercise group and this change was associated with changes in 
PWV; rPearson = 0.27, p = 0.048) as potential confounders, after checking baseline 
group comparisons. As we aimed at assessing efficacy, the primary analyses were defined as 
per-protocol, where patients from the exercise group were included if attendance at the 
exercise sessions was 75%. To assess the robustness of the results, subsequent sensitivity 
analyses (i.e., baseline observation carried forward (BOCF) imputation; per-protocol with 
minimum attendance of 90%, and complete-case analyses) were conducted. A blinded 
investigator (AS-M) undertook the data handling and all hypothesis testing. All the 

analyses were conducted with Stata v.13.1 (StataCorp LP., College Station, TX, USA). 
Statistical significance was set at p < 0.05. 
 
Results 
There were no between-group differences in the changes in arterial stiffness (median 
PWV difference 􀀀0.034, 95% CI 􀀀0.42 to 0.36 m/s; p = 0.860) or hsCRP, TNF- 
, IL-6, and MPO (all p > 0.05) at week 12. In comparison to the control group, the exercise 
group significantly increased cardiorespiratory fitness (median difference 2.26 minutes, 
95% CI 0.98 to 3.55; p = 0.001). These results suggest that 12 weeks of progressive 
treadmill aerobic exercise increases cardiorespiratory fitness without exacerbating arterial 
stiffness, inflammation, or oxidative stress in women with SLE.