Exercise with blood flow restriction improves muscle strength and mass while preserving the vascular and microvascular function and structure of older adults

Abstract

BACKGROUND:

Changes in muscle mass, strength, vascular function, oxidative stress, and inflammatory biomarkers were compared in older adults after resistance training (RT) performed with low-intensity without blood flow restriction (RT-CON); low-intensity with BFR (RT-BFR); and high-intensity without BFR (RT-HI).

METHODS:

Thirty-two untrained individuals (72±7 y) performed a 12-week RT after being randomized into three groups: RT-CON –30% of 1 repetition maximum (RM); RT-BFR –30% of 1RM and mild BFR (50% of arterial occlusion pressure); RT-HI –70% of 1 RM.

RESULTS:

Improvements in handgrip strength were similar in RT-BFR (17%) and RT-HI (16%) vs. RT-CON (–0.1%), but increases in muscle mass (6% vs. 2% and –1%) and IGF-1 (2% vs. –0.1% and –1.5%) were greater (p < 0.05) in RT-BFR vs. RT-HI and RT-CON. Changes in vascular function, morphology, inflammation, and oxidative stress were similar between groups, except for time to reach maximum red blood cell velocity which showed a greater reduction (p < 0.05) in RT-BFR (–55%) vs. RT-HI (–11%) and RT-CON (–4%).

CONCLUSION:

RT with low intensity and mild BFR improved muscle strength and mass in older individuals while preserving vascular function. This modality should be considered an adjuvant strategy to improve muscle function in older individuals with poor tolerance to high loads.

Keywords

Aging resistance training oxidative stress endothelial function

1 Introduction

The aging process is characterized by reductions in muscle mass and strength, compromising physical function and autonomy [1]. Physical training is acknowledged to counteract these losses [2]. For this reason, professional organizations worldwide suggest that resistance training (RT) performed with loads greater than 70% of one-repetition maximum (RM) would be capable to promote positive muscle adaptations in older adults [2].

However, the mechanical stress associated with this RT intensity may be intolerable for individuals with orthopedic fragility [3 –5]. Alternatively, prior studies suggested that RT performed with relatively low intensity (20–30% of 1 RM) combined with blood flow restriction (BFR) would be effective to increase muscle mass and strength [6]. Although the physiological mechanisms underlying these adaptations remain unclear [7], accumulated evidence suggests potential clinical applications of this modality in individuals showing intolerance to high loads [8, 9].

Trials combining RT and BFR that succeeded to improve muscle mass or function usually applied high occlusion pressures [5 , 11]. However, there are concerns about the cardiovascular impact of BFR in older adults, particularly on vascular function [12]. Recent studies showed that higher levels of cuff pressure may increase the retrograde shear rate and consequently the release of pro-atherogenic factors in the endothelium [13, 14]. Decreased flow-mediated dilation (FMD) in the brachial artery [13] and greater release of cellular markers of endothelial activation and apoptosis (e.g. CD62E+ and CD31+/CD42b–) have been documented due to BFR in young subjects [14]. On the other hand, at least one trial showed that further impairments after acute elevation in retrograde shear stress did not occur in older adults with endothelial dysfunction [15]. By accepting the premise that the endothelium of those individuals would be less responsive to shear stress, there is room to speculate that low levels of BFR would not be deleterious. Moreover, the absence of negative effects due to retrograde blood flow is suggestive that a combination of low BFR levels and physical training would provoke favorable vascular adaptations in older individuals.

In short, although prior research has demonstrated that acute exercise performed with BFR may cause endothelial damage in younger individuals [16, 17], additional studies are warranted on the effects of chronic BFR training performed with mild occlusion levels, especially in older subjects [18, 19]. Given this gap in the literature, the present trial compared the effects of RT performed with and without BFR on the muscle mass and strength of upper and lower limbs, microcirculation, vascular function, inflammatory biomarkers, and oxidative stress in older adults. We hypothesized that a 12-week RT performed with low intensity and mild BFR occlusion would be as effective as traditional RT performed with high intensity to improve muscular, vascular, and inflammatory outcomes.

2 Methods

2.1 Participants

Thirty-two healthy and untrained individuals (≥65-yrs-old, 72±7 years) enrolled in the experiment (22 females/10 males). The initial screening included clinical history, physical examination, treadmill exercise testing, and vascular Doppler to assess the atherosclerotic burden of arteries. Exclusion criteria were engagement in moderate or high-intensity physical training in the previous 3 months; kidney, liver, or autoimmune diseases; cancer; severe anemia; severe visual and audition limitations; neurological or orthopedic disabilities; musculoskeletal disorders impairing exercise performance; mini-mental state exam score≤13 pts; obesity; chronic obstructive pulmonary disease; uncontrolled hypertension; unstable angina; recent myocardial infarction, stroke or peripheral vascular disease (< 6 months); chronic heart failure and smoking.

2.2 Recruitment

Volunteers were recruited at the Rio de Janeiro section of the Frailty Study in Brazilian Elderly (n = 284), the Elderly Care ambulatory of the Piquet Carneiro Clinic (n = 71), and the Laboratory of Physical Activity and Health Promotion at the University of Rio de Janeiro State (n = 73). Of the 37 individuals considered eligible, five presented musculoskeletal impairments during the HI protocol (about 19%), and 32 concluded the experiment. Figure 1 presents the flowchart of the enrollment, allocation, and follow-up of participants.

Fig. 1

Flowchart of the study. FIBRA-RJ –Rio de Janeiro Section of the Study Frailty in Brazilian Elderly; CIPI –Care for the Elderly; LABSAU –Laboratory of Physical Activity and Health Promotion.

2.3 Experimental design

Assessments were performed before and after RT (Fig. 2). A detailed explanation of the potential benefits and risks of the research was provided before participants signed informed consent forms. This trial was registered at the Thai Clinical Trials Registry office (TCTR20170131001) and approved by the local Ethics Committee (CAAE: 17782513.0.0000.5282).

Fig. 2

Experimental design.

2.4 Randomization

Eligible volunteers were randomized into three groups: a) Low-intensity control (RT-CON) –exercise intensity corresponding to 30% of 1 RM without BFR; b) Low-intensity BFR (RT-BFR) –exercise intensity corresponding to 30% of 1 RM with BFR equivalent to 50% of arterial occlusion pressure at rest (rAOP); and c) high intensity (RT-HI) –exercise intensity corresponding to 70% of 1 RM without BFR. By including a control group performing low-intensity RT, we aimed to verify whether potential adaptations in RT-BFR could be effectively attributed to the mild blood restriction.

2.5 Resistance training program

The exercise sessions included: a) 15 min of warm-up on a treadmill with intensity corresponding to 30% of heart rate reserve (HRR); b) 30-min of exercises for upper and lower limbs in the following order: elbow flexion, leg press, pulley elbow extension, and knee extension. The individuals performed 3 sets of 10 repetitions with loads corresponding to 30 or 70% of 1 RM (RT-BFR and RT-HI, respectively), interspersed with 1-min intervals between sets and exercises; c) 5-min cool-down with low-intensity stretching exercises. Training sessions occurred from 7 to 10 am, 3 times a week, for 12 weeks. After each exercise session, protein supplementation was provided by a certified nutritionist (15 g dissolved in 150 ml water, Isofort Whey Protein Isolate, Vitafor Nutritional Supplements, Sao Paulo, SP, Brazil). The participants were instructed to keep their physical activity and nutritional habits during the experiment.

2.6 One-repetition maximum test

Participants performed a warm-up set of 8 repetitions with loads corresponding to 50% of their estimated 1RM obtained during the familiarization session. After familiarization, participants remained at rest for 3-min and then performed a maximum of five attempts to achieve their 1RM, with 3-min intervals between attempts. The maximum load on each exercise was recorded following available recommendations [20].

2.7 Blood flow restriction

The BFR was applied using a nylon cuff size 11×85 cm connected to a pneumatic cuff (Hokanson model TD312, Bellevue, WA, USA) placed on the proximal third of the arms and legs and inflated about 50% of rAOP [3, 21]. The cuff was inflated until the blood flow ceased, as assessed by a portable vascular Doppler (DV 610B Medmega^TM, Franca, SP, Brazil). During BFR training, the same relative pressure was applied to the upper and lower limbs. Since exercises were alternated for segments, the cuffs placed on the arm and leg were maintained throughout all sets, including the resting intervals. On average, the BFR during sets lasted 3 min. Interruptions in blood occlusion were allowed during the transition between exercises (60 s), with arm deflating during leg exercises and vice-versa.

2.8 Body composition

Body composition was assessed through dual X-ray absorptiometry (DXA) (DPX-IQ, Lunar Radiation Corporation^TM, Madison, WI, USA) using specific software for whole-body analysis (enCORE Software Platform version 12.20, Chalfont St. Giles, United Kingdom). Fat percentage and mass, lean mass, skeletal muscle mass (SMM), fat-free mass, and bone mineral content were recorded. Appendicular muscle mass (ASM) was calculated as the sum of muscle mass in arms and legs. The scans were performed in high resolution and analyzed by the same trained technician [22].

2.9 Handgrip and isokinetic strength

A hand dynamometer (Model PC 5030J1, Fred Sammons^TM Inc., Burr Ridge, IL, USA) was used to measure handgrip strength (HGS), following recommendations published elsewhere [23]. Volunteers remained seated in a straight-backed chair to measure the HGS in the dominant hand. They were oriented to keep the forearm supinated and were verbally encouraged to perform the maximal effort in three trials interspersed with 2-intervals. Volunteers remained seated in a straight-backed chair with the forearm supinated and performed the maximal effort in three trials interspersed with 2-intervals.

An isokinetic dynamometer (Biodex System 4 Pro, Biodex Medical Systems^TM, Shirley, NY, USA) was used to assess the peak torque of knee extensors. Before the test, participants performed a 5-min warm-up on a cycle ergometer (Cateye EC-1600, Cateye, Tokyo, Japan) with no load and comfortable speed, followed by a familiarization exercise in the dynamometer (one set of 15 repetitions with angular velocity fixed at 120°/s). The isokinetic protocol involved concentric-concentric muscle actions performed by the dominant limb. The range of motion varied between 0° and 90° with the speed fixed at 60°/s. Subjects were encouraged to perform maximal effort during 3 sets of 10 repetitions interspersed with 120-s intervals. The test was considered valid when the coefficient of variation between sets was lower than 15%. Protocol and procedures were administered according to previous studies evaluating the isokinetic strength in older adults [24].

2.10 Venous occlusion plethysmography (VOP) and nailfold videocapillaroscopy (NVC)

Forearm blood flow (FBF) was evaluated by VOP (Hokanson^TM AI6, Bellevue, WA, USA) [21]. Assessments occurred after 6-h fasting (7–10 a.m), in a quiet temperature-controlled room (20–22°C), after 30-min acclimatization, in a supine position. The FBF was measured in the non-dominant forearm kept above the heart level, with a mercury-in-silastic strain gauge placed on the upper third of the forearm (maximum circumference). Measurements included the following phases: FBF at baseline 1; peak FBF during reactive hyperemia, after 5 min of brachial artery occlusion with pressure 50 mmHg above systolic blood pressure (SBP); FBF at baseline 2; and peak FBF after 5 min of 0.4 mg sublingual nitroglycerin administration (Nitrolingual Burns Adler Pharmaceuticals^TM Inc, Charlotte, NC, USA). The measurement duration in each phase was 2 min followed by 3-min intervals to avoid interference in the next phase, except between reactive hyperemia and FBF baseline 2, when a 15-min interval was applied. The FBF during reactive hyperemia was assessed to estimate vasoreactivity as a surrogate of endothelial function. Nitroglycerin has been used to access endothelial-independent vasodilatation.

Microvascular function and morphology were evaluated by NVC [21, 25]. Assessments also occurred in the morning after 6-h fasting, following 30-min acclimatization in a temperature-controlled room (24±1°C). The following outcomes were assessed in the fourth left finger (medial, central, and lateral microscopic regions): functional capillary density (FCD), which is the number of capillaries/unit tissue area with flowing red blood cells with 250x magnification; afferent (AFD), apical (APD) and efferent (EFD) capillary diameters, and baseline red blood cell velocity (RBCVbas). After 1 min of ischemia with cuff pressure above SBP measured on the fourth left finger, the maximum RCDV value (RBCVmax) with 680x magnification, and time to reach RBCVmax (TRBCVmax) during post-reactive hyperemia were registered. The RBCVmax/RBCVbas ratio was determined to quantify the vasodilatation vs. baseline condition. A single trained technician performed all assessments (intra-observer measurement variation –8%).

2.11 Blood biomarkers

After an overnight 8-h fasting, venous blood samples were collected into plasma EDTA tubes to determine soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-6 (IL-6), endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) circulating levels using Human Quantikine ELISA kits (R&D Systems^TM, Minneapolis, MN, USA) and IGF-1, 2 magnetic bead panel (EMD Millipore Corporation^TM, Burlington, MA, USA), for IGF-1 analysis. Blood was also harvested into serum tubes for determination of oxidized low-density lipoprotein (oxLDL), tumor necrosis factor α (TNF-α), and high sensitivity C-reactive protein (hs-CRP) concentrations, using Mercodia Oxidized LDL ELISA (Mercodia^TM, Uppsala, Sweden), Human Quantikine ELISA kit (R&D Systems^TM, Minneapolis, MN, USA) and latex turbidimetric kit (Biosystems^TM S.A., Barcelona, Spain), respectively. All assays were performed according to the kit manufacturer’s instructions.

2.12 Statistical analyses

Data normality was tested by the Shapiro Wilk test and results were expressed as mean±standard deviation (SD), unless stated otherwise. Baseline demographic and clinical characteristics among groups were compared through 1-way ANOVA, and categorical variables by the chi-square test. Heteroscedasticity was tested assuring the homogeneity of variance. Comparisons between groups at baseline were made through 1-way ANOVA.

Considering that our main hypothesis referred to differences between groups and given the risk of type II errors due to the relatively small sample, pre vs. post variations (deltas) were compared through 1-way ANOVA. A post hoc statistical power of 0.85 was obtained using the G-Power software (University of Düsseldorf, Germany) for this statistical approach (between-group comparisons, α err prob: 0.05; effect size: 0.5). Cohen’s d effect sizes were calculated for significant differences. All calculations were performed using the NCSS statistical software (LLCTM, Kaysville, UT, USA), and the significance level was set at p≤0.05.

3 Results

No clinical events occurred during the experiment, and participants allocated in RT-BFR did not report discomfort or pain during the training sessions. In RT-HI, five participants (33%) discontinued the intervention as a consequence of musculoskeletal injuries (see Fig. 1), and 10 concluded the exercise protocol. The average resting systolic and diastolic blood pressure were 130±11 and 76±11 mmHg, respectively. Blood restriction pressures corresponding to 50% of rAOP were 65±5 mmHg. Demographic, medication, and clinical characteristics were similar across groups (Table 1).

Table 1
Demographic characteristics, medications, and clinical history of the experimental groups

Variable RT-CON (n = 10) RT-BFR (n = 12) RT-HI (n = 10) p- value

Demographic characteristics

Age (yrs) 72±8 71±6 73±7 0.70

Weight (kg) 60.9±9.6 65.9±10.5 64.8±11.9 0.54

Height (cm) 160.3±9.2 158.2±10.5 157.9±9.7 0.84

Body mass index (kg/m2) 23.8±2.4 26.3±2.4 25.8±2.9 0.08

Female (%) 6 (60) 8 (66.7) 8 (80) 0.61

Medications –n (%)

Diuretic 1 (10) 2 (16.7) 2 (20) 0.82

Beta-blocker 2 (20) 1 (8.3) 0 (0) 0.30

Angiotensin converting enzyme inhibitor 4 (40) 4 (33.3) 4 (40) 0.93

Anticoagulant 1 (10) 1 (8.3) 1 (10) 0.98

Anti-arrhtymic 0 (0) 0 (0) 1 (10) 0.32

Metformin 2 (20) 2 (16.7) 2 (20) 0.97

Statins 2 (20) 1 (8.3) 2 (20) 0.67

Clinical history –n (%)

Diabetes Mellitus 2 (20) 2 (16.7) 2 (20) 0.97

Hypertension 5 (50) 6 (50) 5 (50) 1

Dyslipidemia 2 (20) 2 (16.7) 2 (20) 0.97

Venous failure 1 (10) 1 (8.3) 1 (10) 0.98

Variable	RT-CON (n = 10)	RT-BFR (n = 12)	RT-HI (n = 10)	p- value
Demographic characteristics
Age (yrs)	72±8	71±6	73±7	0.70
Weight (kg)	60.9±9.6	65.9±10.5	64.8±11.9	0.54
Height (cm)	160.3±9.2	158.2±10.5	157.9±9.7	0.84
Body mass index (kg/m2)	23.8±2.4	26.3±2.4	25.8±2.9	0.08
Female (%)	6 (60)	8 (66.7)	8 (80)	0.61
Medications –n (%)
Diuretic	1 (10)	2 (16.7)	2 (20)	0.82
Beta-blocker	2 (20)	1 (8.3)	0 (0)	0.30
Angiotensin converting enzyme inhibitor	4 (40)	4 (33.3)	4 (40)	0.93
Anticoagulant	1 (10)	1 (8.3)	1 (10)	0.98
Anti-arrhtymic	0 (0)	0 (0)	1 (10)	0.32
Metformin	2 (20)	2 (16.7)	2 (20)	0.97
Statins	2 (20)	1 (8.3)	2 (20)	0.67
Clinical history –n (%)
Diabetes Mellitus	2 (20)	2 (16.7)	2 (20)	0.97
Hypertension	5 (50)	6 (50)	5 (50)	1
Dyslipidemia	2 (20)	2 (16.7)	2 (20)	0.97
Venous failure	1 (10)	1 (8.3)	1 (10)	0.98

RT-CON –control; RT-BFR –blood flow restriction; RT-HI –high intensity; p-value –alpha levels from the ANOVA or Chi-square Test; results expressed as mean±standard deviation or absolute (relative frequency).

No differences between groups were found at baseline for most of the observed outcomes (body composition and strength, vascular function, microcirculation, and blood biomarkers). The exceptions were IL-6 (RT-BFR > RT-CON and RT-HI; p < 0.05) and RBCVmax/RBCVbas (RT-CON > RT-HI, p < 0.05). Table 2 exhibits data on changes in body composition and strength. Increases in ASM and SMM were greater in RT-BFR than RT-CON. Variations in peak torque and total work extension were similar between groups. On the other hand, increases in HGS were greater in RT-HI and RT-BFR vs. RT-CON.

Table 2

Outcomes of body composition and strength at baseline and deltas in experimental groups

Variable	RT-CON (n = 10)			RT-BFR (n = 12)			RT-HI (n = 10)
	Before	Δ post-pre intervention	Effect size	Before	Δ post-pre intervention	Effect size	Before	Δ post-pre intervention	Effect size	p-value
Body composition
Fat percentage (%)	35.4±8.5	–0.23±1.49	0.02	38.4±6.8	–0.96±2.11	0.13	37.8±6.2	–0.49±0.86	0.09	0.57
Fat mass (kg)	20.69±5.61	–0.00±1.23	0	24.75±6.57	–0.56±1.20	0.08	24.19±6.95	–0.06±0.70	0.008	0.42
Lean mass (kg)	38.08±8.19	0.18±0.84	0.02	38.82±6.85	1.37±3.05	0.19	39.41±7.42	0.73±1.15	0.09	0.40
Fat free mass (kg)	40.23±8.64	0.23±0.80	0.02	41.97±8.65	–0.29±1.64	0.03	41.40±7.87	0.64±1.51	0.07	0.29
Bone mineral content (kg)	2.15±0.46	–0.00±1.19	0	2.29±0.68	0.04±0.13	0.05	1.99±0.46	–0.007±1.966	0.02	0.30
Lean mass (kg)
ASM	16.67±4.37	–0.10±0.36	0.02	17.75±4.97	0.87±1.30^*	0.18	16.90±3.70	0.53±0.78	0.13	0.05
SMM	18.60±5.13	–0.12±0.41	0.02	19.81±5.87	0.99±1.47^*	0.18	18.75±4.44	0.60±0.88	0.13	0.05
Strength muscle
Hand grip strength (kgf)	22.4±7.9	–0.02±0.71	0	27.2±9.0	4.65±1.31^*	0.52	21.5±6.4	3.49±2.24^*	0.54	< 0.001
Peak torque extension (nm)	81.1±18.3	3.21±10.64	0.14	105.0±37.5	10.17±17.35	0.37	86.6±22.9	11.36±5.32	0.50	0.43
Total work extension (j)	699.6±109.9	21.6±77.1	0.15	874.6±312.2	26.15±172.47	0.08	863.8±287.1	59.29±131.29	0.21	0.83

RT-CON –control; RT-BFR –blood flow restriction; RT-HI –high intensity; ASM –appendicular skeletal muscle mass; SMM –skeletal muscle mass; ^*: p < 0.05 for comparisons between RT-HI vs. RT-CON and RT-BFR vs. RT-CON; results expressed as mean±standard deviation.

Inter-individual coefficients of variation for outcomes assessed using VOP and NVC ranged from 10–15% and 10–20%, respectively. Table 3 presents the changes in variables reflecting vascular function, microcirculation, and blood biomarkers. Decreases in TRBCVmax and increases in IGF-1 were greater in RT-BFR vs. RT-CON and RT-HI.

Table 3

Outcomes of vascular function, microcirculation, and blood biomarkers at baseline and deltas in experimental groups

Variable	RT-CON (n = 10)			RT-BFR (n = 12)			RT-HI (n = 10)
	Before	Δ post-pre intervention	Effect size	Before	Δ post-pre intervention	Effect size	Before	Δ post-pre intervention	Effect size	p-value
Vascular function
FBF Baseline 1 (ml/min/100 ml)	1.48±0.74	0.2±0.9	0.37	2.16±1.18	0.1±1.2	0.13	2.25±1.06	–0.1±0.5	0.18	0.57
FBF Hyperemia (ml/min/100 ml)	9.91±3.51	4.5±5.7	0.81	13.51±5.44	3.1±6.0	0.50	10.47±4.07	2.2±3.9	0.58	0.62
FBF Baseline 2 (ml/min/100 ml)	1.23±0.55	0.4±0.9	0.55	1.68±0.91	0.6±0.8	0.68	1.88±0.79	0.1±0.9	0.13	0.38
FBF Nitroglycerine (ml/min/100 ml)	1.92±0.73	0.2±1.1	0.31	1.93±0.61	0.3±1.0	0.51	2.69±1.53	0.5±2.3	0.31	0.94
Microvascular morphologic
AFD (μm)	8.7±1.3	0.10±4.82	0.06	10.0±3.4	–0.84±5.13	0.45	10.5±6.2	1.02±7.42	0.28	0.78
APD (μm)	14.8±3.2	–0.28±8.36	0.09	15.3±4.2	–2.5±10.79	0.72	19.0±11.3	–1.11±14.88	0.67	0.91
EFD (μm)	13.9±2.0	1.46±10.06	0.38	15.2±3.6	–1.25±7.71	0.58	16.5±5.0	1.32±11.17	0.57	0.78
Microvascular function
FCD (n/mm2)	22.8±10.0	4.47±21.76	0.64	22.9±17.0	–1.60±13.39	0.30	29.9±16.2	–3.14±22.20	0.23	0.59
TRBCVmax(s)	5.00±1.87	–0.20±3.61	0.11	7.40±3.71	–4.10±4.68^*	1.19	5.57±1.67	–0.60±4.11^φ	0.74	0.03
RBCVmax/RBCVbas (%)	1.26±0.12	0.02±0.68	0.07	1.18±0.11	0.05±0.53	1.03	1.09±0.07	0.33±0.73	1.05	0.18
Blood biomarkers
hs-CRP (mg/dl)	0.08 [0.03–0.15]	0.07±0.15	0.43	0.24 [0.08–0.68]	–0.25±0.75	0.45	0.15±0.11	0.05±6.47	0.43	0.19
TNF-α (pg/ml)	1.18 [0.99–1.36]	6.54±0.13	0.24	1.27±0.25	–1.77±0.23	0.06	1.26 [1.21–1.39]	–9.69±0.27	0.31	0.29
IL-6 (pg/ml)	1.59±0.60	0.34±0.82	0.44	3.61±3.00	–1.12±3.50	0.43	1.82±0.86	0.36±1.68	0.30	0.25
sVCAM-1 (ng/ml)	913.2±174.3	–6.51±78.76	0.03	796.7±143.4	11.16±63.07	0.08	897.6±195.8	–26.28±80.18	0.13	0.50
sICAM-1 (ng/ml)	202.2±80.2	–1.34±20.76	0.01	183.3±37.3	–7.91±12.87	0.21	209.8±79.5	–2.80±14.46	0.03	0.60
ET-1(pg/ml)	1.50±0.52	2.33±0.46	0.06	1.43±0.31	2.78±0.22	0.09	1.63±0.48	–5.28±0.55	0.10	0.88
oxLDL (u/l)	57.03±20.19	0.85±13.80	0.04	64.60±10.52	0.36±12.39	0.02	60.17±18.95	–1.96±11.92	0.11	0.86
IGF-1 (ng/ml)	46.19±13.13	–6.91±9.92	0.58	39.67±23.16	9.20±9.19^*	0.52	40.60±19.91	–3.79±10.06^φ	0.20	0.001

RT-CON –Control; RT-BFR –blood flow restriction; RT-HI –high intensity; FBF –forearm blood flow; bas –baseline; hyper –hyperemia; nitro –nitroglycerine; AFD –afferent capillary diameters; APD –apical capillary diameters; EFD –efferent capillary diameters; FCD –functional capillary density; TRBCVmax –time to reach RBCVmax; RBCVmax/RBCVbas –increment of maximal red blood cell velocity (RBCVmax) from baseline (RBCVbas); CRP –c-reactive protein; TNF-α –tumoral necrosis factor-alpha; IL-6 –interleukin-6; sVCAM-1 –soluble vascular cell adhesion molecules; sICAM-1 –soluble intercellular adhesion molecules; ET-1 –Endothelin-1; oxLDL –oxidized low-density lipoprotein; IGF-1 –insulin-like growth factor 1; ^*: p < 0.05 for the comparison between RT-BFR vs. RT-CON; ^φ: p < 0.05 for the comparison between RT-BFR vs. RT-HI; results expressed as mean (standard deviation) or median [quartiles].

4 Discussion

The present study demonstrated that RT-BFR performed with mild occlusion level (50% of rAOP) was effective to increase muscle strength (HGS) and mass (ASM and SMM), and improving biomarkers related to hypertrophy (IGF-1) vs. RT-HI and RT-CON in older individuals. Moreover, improvement in microvascular function (reflected by lower TRBCVmax) during reactive hyperemia was observed in RT-BFR, but not RT-HI or RT-CON. Changes in the integrity of smooth muscle cells (reflected by endothelial independent vasodilatation), microvascular morphological (AFD, APD, and EFD) and functional variables (FCD and RBCVmax/RBCVbas), inflammatory profile (hs-CRP, TNF-α and, IL-6), endothelial injury biomarkers (sICAM-1, sVCAM-1 and ET-1), and oxidative stress (oxLDL) were similar between groups. To the best of our knowledge, our study is the first to demonstrate that RT performed with a low BFR level is capable to promote benefits on the microvascular function of older individuals, along with improvements in appendicular muscle mass and strength.

Previous studies have shown the effectiveness of RT performed with BFR to preserve muscle strength and mass in older individuals [5 , 10]. However, those trials generally applied more restrictive BFR than ours, ranging from 160- to 200 mmHg [5, 10]. We applied a substantially lower blood occlusion pressure since prior research suggested an inverse relationship between the restriction level and changes in endothelial function [13].

Although the available evidence indicates that BFR training with high occlusion levels is capable to increase muscle strength and mass after short periods of intervention (4–6 weeks) [10, 26], few studies reported data on muscle adaptations in older individuals due to RT combined with low BFR levels [3 , 27]. We hypothesized that RT performed with low-intensity and BFR with cuff pressures lower than rAOP might induce similar gains vs. high-intensity RT. In this case, improvements in muscle function would occur with a minor risk of endothelial harm. Our findings confirmed this hypothesis since favorable adaptations occurred in muscle strength and mass. Increases in muscle strength and mass were similar to those observed by experiments applying training protocols with similar intensity and volume [3, 27].

The relatively short duration of the RT interventions may help to justify why ASM and SMM increased in RT-BFR but not RT-HI vs. RT-CON [2]. Although merely speculative, this premise is reinforced by the fact that an increase in IGF-1 was detected only in RT-BFR. The IGF-1 is acknowledged to have anabolic effects influencing muscle adaptations to exercise [28], and our data confirm prior studies showing IGF-1 increasing after RT performed with BFR vs. traditional high-load RT [29 –31]. This suggests that even when the occlusion pressure is mild, BFR may optimize hypertrophy pathways in older adults in comparison with training alone. In short, the improvements in strength, ASM, and SMM along with the increased IGF-1 allow thinking that a more positive drive for hypertrophy occurred in RT-BFR vs. RT-HI [32]. Although promising, these results shall be interpreted with caution. The DXA is not a direct measure of muscle mass, and small effect sizes were observed for ASM in both RT-HI and RT-BFR. Trials with longer training durations and more specific assessments of muscle mass would be necessary to clarify this issue.

It is widely accepted that decreases in muscle mass and strength predispose to motor disability among older adults [1], and RT to counteract those reductions is recommended. However, muscle hypertrophy relies on an optimal combination of high loads and volume [2], which is not always feasible in older populations due to injury risk [33]. Moreover, high-intensity RT may be uncomfortable, which compromises the training adherence [34]. In this context, the fact that a relatively short RT performed with light workloads might induce gains in muscle strength and mass when combined with mild BFR represents useful information for practitioners. Those promising results warrant further research.

There are many techniques to assess microcirculation or peripheral vascular function [35]. VOP measures the FBF in the brachial artery based on forearm volume changes, being applied to assess the peripheral vascular function [36]. The NVC evaluates the structure and function of microcirculation. VOP and NVC may be acknowledged as complementary. The VOP assesses microcirculation indirectly through brachial artery vasoreactivity. Through ischemia stimuli and the use of sublingual nitroglycerin, we produced vasodilation with increased blood flow, which is functional analysis. On the other hand, NVC directly evaluated the structure of microcirculation through capillary diameters and morphology, and microvascular function through FCD, RBCVmax, and TRBCVmax [37].

There is extensive evidence of the positive effects of RT to counteract vascular dysfunction in older individuals with or without cardiovascular diseases [38, 39]. Exercise training with BFR has been suggested to compromise the endothelial function due to retrograde blood flow, which seems to stimulate pro-atherogenic phenotypes of endothelial cells [14 , 40]. The influence of the vascular occlusion magnitude in this context is yet unknown. To test whether RT performed with low intensity and mild BFR levels damaged microcirculation, we have assessed the vascular reactivity during reactive hyperemia and endothelium-independent vasodilation. A greater decrease in TRBCVmax occurred in RT-BFR vs. RT-HI or RT-CON. Multiple factors not related to the endothelium concur for the responses due to reactive hyperemia, such as myogenic relaxation, nitric oxide, adenosine, or prostaglandins [41 –43]. Unfortunately, our data are also insufficient to determine the specific mechanisms underlying this potential improvement in vascular function. Acute elevations in retrograde shear stress seem not to provoke further impairments in older individuals with already compromised endothelial function [15]. Thus, shear stress alone cannot explain the increased vasoreactivity presently observed. We did not evaluate the impact of retrograde blood flow on vascular function, since we assumed that retrograde flow was similarly increased throughout.

The FBF during hyperemia increased after both RT-BFR and RT-HI, but changes were similar vs. RT-CON. Improvements in vascular function have been reported after RT with different intensities [44]. Our findings partially concur with this premise, since slight and equivalent increases in vasodilation response occurred in all groups. However, the absence of a nonexercise control group limits the possibility of making inferences about the relative effectiveness of RT interventions upon each other. The most important implication of our results does not lie in the potential improvement in vasodilation after the RT interventions, but in the fact that worsening was not found in any of them. The comparison between groups made it quite clear that changes in FBF hyperemia were not different between RT-BFR vs. RT performed without blood occlusion regardless of the load intensity. This reinforces the hypothesis that BRT training with moderate occlusion would not be harmful to vascular function in older individuals.

It is worthy to notice that some studies showed acute reductions of flow-mediated vasodilatation after BFR exercise in young individuals [16, 17], but not in older groups [15]. This age-related difference in endothelial responses has been also found in the few trials that investigated the chronic vascular adaptations to BFR training [19, 45]. Therefore, acute and chronic vascular adaptations to retrograde shear rates may be different in young and older adults. This premise concurs with our findings and should be taken into account when designing RT for older adults, which often exhibit poor endothelial function [3, 7]. Although RT appears to promote beneficial effects in endothelial function [46], high loads should be applied with caution [47].

Changes in capillary diameters and FCD were similar between groups after the interventions, indicating that the microcirculatory morphology and tissue perfusion were preserved. It is worthy to notice that at baseline the RBCVmax/RBCVbas ratio was slightly higher in RT-CON than in RT-HI (< 10%). However, values in RT-BFR and RT-HI were similar, and at the end of the interventions, all groups showed similar variation. The reduction in TRBCVmax was greater in RT-BFR vs. RT-HI and RT-CON. The NVC technique evaluates capillary dynamics and reflects the influence of arterioles in microcirculation. A decreased time to reach RBCVmax indicates a good response of microcirculation to ischemia. Previous data from our group suggested an age-related decrease in vasoreactivity among older adults [48]. The fact that the capillary diameter and perfusion (FCD) were preserved in RT-BFR is therefore positive.

Finally, changes in inflammation status (hs-CRP, TNF-α, IL-6, and hs-CRP), oxidative stress (oxLDL), and endothelial injury (sICAM-1, sVCAM-1, and ET-1) were similar across groups. Older individuals often exhibit higher inflammation and oxidative stress than young adults [47, 49]. There is evidence acknowledging the protective effects of physical training against age-related increases in oxidative stress and chronic inflammation [50], and at least one trial showed that RT performed with low BFR reduced the production of reactive oxygen species in older adults [51]. Those premises could not be confirmed in the present study. Unfortunately, the data dispersion probably precluded the detection of differences between groups in those outcomes, even when absolute differences seemed to be elevated (please, refer to Table 3). On the other hand, our data support that RT performed with mild BFR and traditional RT protocols would not induce different changes in inflammatory or oxidative stress biomarkers in older adults. Further research is warranted to ratify those findings and clarify the potential impact of BFR training on inflammation and oxidative stress in this population.

The present study has limitations. Firstly, retrograde blood flow was not measured. However, it is important to notice that the blood flow pattern was not a dependent variable in our study. Measuring blood flow would certainly help to explain our results, reinforcing or not the hypothesis that there would be a dose-response relationship between RT-BFR and retrograde blood flow [13 , 52]. However, the vascular response to reactive hyperemia is not only necessarily determined by changes in shear rate. It would be necessary to determine whether shear stress and vasoreactivity are correlated, as well as the relative role of other mechanisms such as oxidative stress or neural response. Nutritional intake and physical activity levels during the study were not assessed, but the participants followed dietary recommendations for older adults [53] and were instructed to avoid changes in their physical activity patterns. Another potential source of bias is the gender influence on the responses to BFR training. Unfortunately, the small sample precluded analyzes controlling for this factor. Even though the intensity corresponding to 30% of 1 RM applied in RT-CON was probably insufficient to increase muscle strength and mass [54], it must be acknowledged that the absence of a “non-exercising” control group is a limitation of our design. Finally, the training volume was not matched across groups. However, previous studies reported equivalent gains in muscle strength and mass after similar periods of RT performed with BFR, irrespective of the training volume [55, 56].

5 Conclusion

In conclusion, RT performed with low intensity and mild BFR occlusion induced equivalent gains in the handgrip and knee-extensors isokinetic strength, but greater increases in the muscle mass of lower and upper limbs in comparison with traditional RT performed with high intensity. Increased levels of IGF-1 occurred only in RT-BFR, suggesting more favorable adaptations in hypertrophy pathways in comparison with RT-HI. Overall, changes in markers of vascular function (vasodilation and endothelial injury) were similar in both RT-BFR and RT-HI. The vascular and endothelial function seemed to be preserved when RT was performed with low-occlusion BFR, which probably attenuated the potentially deleterious effects of retrograde blood flow.

In practical terms, BFR performed with mild occlusion pressures could be included as a safe adjuvant strategy to improve or preserve the strength and muscle mass in older individuals with poor tolerance to high loads. Further research including larger samples of young and older adults, applying longer training durations, performing measurements of the retrograde blood flow, and isolating the role of different mechanisms of vasodilation response would be useful for a better understanding of the hemodynamic impacts of RT performed with BFR in different populations and clinical settings.

Footnotes

Acknowledgments

The authors thank the volunteers and the multidisciplinary team that worked on this project.

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