Patients with stable CAD were recruited from the Center for Preventive Cardiology, Department of Vascular Diseases, Division of Internal Medicine, University Medical Centre Ljubljana, or Coronary Club Ljubljana, both located in Slovenia.

Patients with documented CAD (>3 months after a myocardial infarction, percutaneous coronary intervention or/and coronary artery bypass grafting), aged between 18 to 75 years and physically active more than three times a week were included into the study. All participants were clinically stable and engaged in regular unsupervised physical activity (e.g., walking, cycling) after completion of cardiac rehabilitation, as assessed with short interview. Exclusion criteria were unstable or uncontrolled cardiovascular disease/event (unstable angina, recent myocardial infarction <3 months prior to inclusion, class III or IV heart failure, uncontrolled dysrhythmias, severe pulmonary hypertension, severe and/or symptomatic valve disease, acute myocarditis, endocarditis, or pericarditis, aortic syndrome or venous thromboembolism), acute systemic illness, uncontrolled hypertension (>180/110 mmHg), postural hypotension (≥20 mmHg drop in systolic blood pressure with symptoms of dizziness or light-headedness) (Williams et al., 2007; Wise and Patrick, 2011).

Study was designed as randomized, open-label clinical trial aligned with the CONSORT guidelines (Schulz et al., 2010), adapted for two parallel groups (Figure 1). Participants were randomized into 2 groups (1 interventional and 1 control group) with ratio of 1:1 using adaptive (urn) randomization with sealed envelopes and randomization concealment from the recruiting investigator. Measurements were performed two times: at baseline and after the intervention period (8 weeks). Patients in both groups underwent clinical examination prior to inclusion and ultrasonographic assessment [systemic brachial vascular function and muscle thickness of m. vastus lateralis (VL)], muscle strength assessment and blood sample withdrawal at baseline and post-training intervention. The primary outcome of the study was change in muscle strength and hypertrophy from baseline to 8-week follow-up. The secondary outcome was change in vascular function. Other exploratory outcomes included cardiovascular markers, insulin resistance and inflammatory markers.

Measurements and exercise intervention were conducted at the Center for Preventive Cardiology, University Medical Centre Ljubljana, Slovenia. One week ahead of baseline measurement participants were familiarized with testing and training procedures. The intervention period lasted for 8 weeks of BFR resistance training period with baseline and post-training testing.

During the week ahead of study, all subject underwent full medical examination and a familiarization session. Each testing session was split to 2 days, with at least a day of rest between testing. On the first day, ultrasonographic images were taken to assess flow mediated dilatation (FMD) and muscle thickness, and unilateral 1-RM strength was assessed as well. During the second day, blood samples were collected before subjects performed 3 sets of 10 repetitions of BFR resistance exercise at an intensity of 30% of their previously achieved 1-RM leg extension. All subjects performed a standardized warm-up before each testing or training session as noted in previous section.

Prior to inclusion, all patients underwent a run-in phase aerobic exercise training for 4 weeks (3 times a week, 45 min of cycling, walking or combination thereof at 70–80% maximum heart rate). After the baseline measurements, the control group continued with usual care (aerobic exercise training), while in the intervention group BFR training was added on usual care.

Both groups were informed of the risk associated with the methods and procedures, and signed written consent prior to their inclusion. The protocol was approved by the Republic of Slovenia National Medical Ethics Committee and was registered on ClinicalTrials.gov (identifier: NCT03087292). The study was conducted in accordance with the Declaration of Helsinki and the American College of Sports Medicine guidelines for Use of Human Participants.

Subjects in BFR resistance training group trained for 8 weeks, performing a total of 16 unilateral leg extension exercise sessions. During each week two exercise sessions were performed with 48 h of rest period in between. Each training sessions consisted of three sets of 8, 10, and 12 repetitions in first, second, and third set, respectively, with 45 s inter-set rest interval. Training intensity was initially set to 30% of 1-RM. At every next training session the number of repetitions was increased for at least two repetitions per set. Additionally, training load was increased every 2 weeks, from initial 30% 1-RM to 32.5% 1-RM in the third week, to 37.5% 1-RM in the fifth week and finally to 40% 1-RM in the seventh week. Furthermore, as the training intensity increased, the number of repetitions was lowered to those in first training session. A lifting cadence of 1 s:2 s (concentric: eccentric part) was used for both groups throughout the full range of motion. For exercise purposes, BFR was applied by compressing the medium part of each thigh separately using a pneumatic cuff (Riester, Jungingen, Germany). The cuff was 23 cm width and 42–50 cm in length. Before each session or test, the cuff was inflated between 15 and 20 mmHg greater than resting brachial systolic pressure (Manini and Clark, 2009), taking into consideration the width of the cuff (Hackney et al., 2012) and thigh circumference (Loenneke et al., 2012a). Pressure was maintained throughout the entire training session and was released at the end of last set. To assure safety of the patients, brachial blood pressure and heart rate were measured at the rest, after sets and 5 min after the end of each training session. Brachial systolic and diastolic pressures were monitored using automatic blood pressure monitor (Omron M6, Omron Healthcare, Inc., Vernon Hills, IL, United States) and heart rate was obtained using telemetry (Polar, Kempele, Finland). All measurement and training sessions were monitored by cardiologist and kinesiologist.

BFR resistance training and unilateral isotonic leg extension strength measurements were performed on leg extension machine (Technogym, Cesena, Italy) following previous recommendations (Brown and Weir, 2001; Karabulut et al., 2011). Maximal strength was assessed by performing 1-RM testing at baseline (pre-training) and after (post-training) the completion of exercise intervention. The participants were familiarized with exercise testing protocol and were advised with proper lifting technique at least 3 days prior to testing. Patients were advised to perform the exercise while seated in an upright position with their back in permanent contact with the machine during the test, and with hands holding the handles of the machine. Before the measurement, participants were instructed to complete a warm up that included a 5–8 min of brisk walking (>4 km/h) on treadmill followed by two sets of static stretching for quadriceps muscles. During the test, participants were instructed to complete a warm-up set comprised of 8–10 repetitions for each leg at approximately 50% of their perceived maximal effort (1-RM). The weight was then increased progressively each set with simultaneously lowering number of repetitions until reaching the maximum weight that could be lifted for one repetition. Between each maximal effort there was a 2–3 min rest interval. Maximal muscle strength was determined within five attempts. Before and after measurement brachial blood pressure was obtained, while heart rate was monitored throughout the procedure.

Muscle thickness and vascular function was assessed with Aloka Prosound α7 ultrasound machine (Hitachi Healthcare Americas, Twinsburg, OH, United States) at baseline and after the completion of the study. Both measurements were performed at the same exact time of the day, mostly in the mornings.

Muscle thickness was assessed at rest, with subjects performing no physical activity at least a day before the testing. After applying hypoallergenic, watersoluble transmission gel, ultrasound transducer was placed on the surface of the skin. Muscle thicknesses of right VL were assessed. The measurement sites were determined at upper, midway and lower thirds between the greater trochanter and the lateral epicondyle of the knee. Those distances were measured with the subjects standing still with their knees fully extended. Three longitudinal images of the VL were recorded for each measurement, and the mean of the three values was used for further analysis (Martín-Hernández et al., 2013). Images were then independently analyzed by two experienced researchers and the mean of both was used as a final result.

Flow-mediated dilation was measured on the right brachial artery, approximately 5 cm above the antecubital fossa, according to previously described guidelines (Corretti et al., 2002). The participants were instructed to lie in supine position. The artery was firstly visualized in the horizontal position on the screen, after which three measurements of the arterial diameter were obtained (d1). A cuff was then inflated below the antecubital fossa with the pressure of 50 mmHg above the systolic blood pressure. Ischemia was maintained for 4.5 min. Sixty seconds after the cuff deflation, three measurements of the arterial diameter were obtained again (d2). FMD was calculated with the following formula: [mean(d2) - mean(d1)]/mean(d1) and expressed in %.

Blood samples were drawn from the antecubital vein using a 21-gauge needle; firstly, into a 4.5 mL vacuum tube containing 0.11 mol/L sodium citrate (9:1 v/v) (Becton Dickinson, Vacutainer System Europe, Heidelberg, Germany), followed by serum and K3-EDTA vacuum tubes (Laboratory Technic Burnik, Ljubljana, Slovenia). Plasma was prepared with 20-min centrifugation at 2000 ×g and 15°C. Serum was prepared with 20-min centrifugation at 2000 ×g and 20°C. Half milliliter aliquots of serum were snap frozen in liquid nitrogen and stored at ≤-70°C until analysis. Concentration of glucose was measured in fresh serum on the Fusion 5.1 biochemistry analyzer (Ortho Clinical Diagnostics, Rochester, NY, United States). Levels of CD40 ligand, insulin, TNF-α were measured in a thawed serum aliquote with the Luminex’s xMAP^® Technology utilizing magnetic beads coupled with specific antibodies, which allowed multiplexing. Analysis was performed according to manufacturer’s instructions (R&D Systems, Abingdon, United Kingdom). Homeostatic model assessment (HOMA) was calculated using values plasma glucose levels and insulin levels via the HOMA Calculator¹.

Sample size was calculated based on muscle strength as our primary outcome, as the loss of muscle strength in CAD patients is attributed to long term bed confinement, physical inactivity and in some cases also significant impairment in the cardiovascular disease itself (Bjarnason-Wehrens et al., 2004). The calculations suggested that 28 patients with CAD should be included in order to detect effect size value for muscle strength after BFR training larger 0.58 as described previously (Loenneke et al., 2012b), with an actual power of 0.95 at a level of statistical significance <0.05. Statistical power and sample size was calculated using G^∗Power statistical software (University of Düsseldorf, Germany).

Numeric variables were described as mean values and standard errors of mean, and categorical variables were described as numbers. Data were firstly screened for normality of distribution and homogeneity of variances and/or regression through Shapiro–Wilk’s test, Levene’s test and interaction between independent variables × covariate, respectively. Baseline and post-training differences between groups were determined with the Independent-Samples t-test for normally distributed variables and equal variances between groups, and Mann–Whitney U test was used for asymmetrically distributed variables and/or unequal variances between groups. Two-way analysis of variance (ANOVA) for repeated measurements was used to calculate main effects of group, time and group × time interaction. Within group effect of training intervention (baseline vs. post-training) was assessed with Paired-Samples t-test for normally distributed variables and with Wilcoxon’s test for asymmetrically distributed variables. All data are displayed in the text, tables and figures. The data were analyzed using IBM SPSS Statistics v.21 statistical software package for Windows (SPSS Inc., Chicago, IL, United States). The level of significance was set a priori to an alpha of <0.05.

Post-training responses of heart rate (RR), systolic and diastolic blood pressure following exercise intervention are shown in Table 2. At baseline there was a significantly lower systolic blood pressure in CON group (p = 0.024), with no significant differences in resting heart rate or diastolic blood pressure. After the training intervention, there was a significant main effect for group × time interaction (p = 0.001; η² = 0.627) and trend for group effect (p = 0.074) on systolic blood pressure. Contrary, no significant main effects of time, group or group × time interaction for resting heart rate and diastolic blood pressure were observed. Furthermore, there was a significant decrease in systolic blood pressure in BFR group after training intervention (p = 0.030), whereas similar decrease was not observed in diastolic blood pressure and resting heart rate.

Acute hemodynamic responses to exercise are presented in Table 3. In both groups BFR exercise evoked significant increase in heart rate (p < 0.001) and systolic blood pressure at baseline and post-training exercise test, while diastolic blood pressure did not increase significantly. There was no significant difference between groups in hemodynamic change (at rest vs. post-last set).

At baseline and post-training there were no significant differences between groups in maximal muscle strength (Figure 2). After the training period, there was a significant main effect for time (p < 0.001, η² = 0.769) and group × time interaction (p < 0.001; η² = 0.724), but no for group (p = 0.233). Both groups significantly improved muscle strength post-training, with greater increase in the BFR resistance training group (16.37%, p < 0.001) than control group (5.29%, p < 0.01). Post-training leg strength increased for 8.96 and 2.88 kg in the BFR resistance training group and the control group, respectively.

VL muscle thickness was assessed pre- and post-training period (Table 4). There was no between-group difference in muscle thickness observed at baseline nor after the completion of the study. After the training intervention, there was a significant main effect for time in lower third of VL (p < 0.05; η² = 0.360) alongside borderline significant time effect in midway third of VL (p = 0.072; η² = 0.265), whereas no significant group × time interaction was obtained in neither of all three thirds of VL. Additionally, a significant post-training decrease in muscle thickness of lower VT was observed in CON group (p = 0.031). On the contrary, improvements of muscle thickness on upper and midway thirds of VL in the BFR resistance training group showed a trend toward statistical significance (1.58–1.67 cm, p = 0.096 and 1.60–1.68 cm, p = 0.082, respectively).

Two-way ANOVA showed no significant main effect for group (p = 0.134) or group × time interaction (p = 0.28), whereas a borderline significance was observed for time effect (p = 0.092; η² = 0.236). There were no significant differences between groups or pre- vs. post-training within group, although a trend toward improvement of FMD was obtained in the BFR resistance training group (6.48 ± 0.80% to 8.04 ± 0.98%, p = 0.079, respectively) (Figure 3).

Baseline between group differences were observed in insulin levels and insulin resistance (HOMA IR; both biomarkers p = 0.014; Table 4). After the training intervention, there were significant main effects for time (p = 0.036; η² = 0.340) for CD40 ligand, significant main effect for group (p = 0.005; η² = 0.306) and group × time interaction (p = 0.036; η² = 0.342) for insulin levels. In BFR group, training intervention led to a borderline significant decrease in insulin (p = 0.077), HOMA IR (p = 0.079) and CD40 ligand levels (p = 0.052), while a similar trend was additionally observed in the control group for CD40 ligand levels (p = 0.060).