A total of 140 women from the Saratoga Springs, NY area, responded to emails, flyers, and local newspapers to advertisements regarding the study. A total of 59 subjects were initially screened, of which 30 were eligible for participation (Figure 1
Participants were nonsmoking, healthy, exercise-trained women with no known cardiovascular, renal, or metabolic diseases as assessed by a medical history and a comprehensive medical examination. All participants were highly active (minimum of >30 min, 4 day/week of structured physical activity), lean (BMI < 25 kg/m2; % body fat <30%), middle-aged (25–55 years), and weight stable (±2 kg) for at least 6 months prior to the beginning of the study assessed through questionnaire. All participants provided informed written consent prior to participation, and the study was approved by the Human Subjects Institutional Review Board of Skidmore College (IRB #: 1401-382). All experimental procedures were performed in accordance with the Federal Wide Assurance and related New York State regulations, which are consistent with the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research and in agreement with the Helsinki Declaration as revised in 1983. This study was registered with ClinicalTrials.gov Identifier: NCT02593656.
2.3. Nutrition Intervention
Meal plans were identically matched in terms of total kcals, meal frequency and timing, and dietary support. By design, the only difference between the two groups was the amount of protein (1.0 vs.
2.0 g/kg BW per day). Additional supplementation (daily multi-vitamin/minerals, and caffeine and electrolytes on workout days) was also provided to participants and differed only by the type of product manufacturer. Participants in both groups were provided detailed meal plans designed by a registered dietitian and instructed to follow the meal plans throughout the 12-week intervention (Table S2
). The registered dietitian met with participants weekly for the first two weeks and thereafter on an “as needed” basis. In addition, investigators met with participants a minimum of four days per week to answer questions and reinforce meal plans. To facilitate adherence to the meal plans, food was provided to both groups.
PRISE meal plans included protein-pacing (P; 5–6 meals/day at 2.0 g/kg BW/day) on all days, three of which were whey protein-supplemented (IsaPro®
: 150 kcals, 27 g protein, 3 g carbohydrate, 1.5 g fat; IsaLean Pro®
: 280 kcals, 36 g protein, 21 g carbohydrate, 6 g fat; and IsaLean Bars®
: 210 kcals, 18 g protein, 28 g carbohydrate, 5 g fat—Isagenix LLC, Chandler, AZ, USA). On exercise days, they were supplemented with a caffeine (e+®
: 85 mg caffeine, 8 g carbohydrate) and electrolyte beverage (Replenish®
: 35 kcals, 9 g carbohydrates, 110 mg sodium, 95 mg potassium—Isagenix LLC, Chandler, AZ, USA), and a multi-vitamin/mineral (Ageless Essentials®
—Isagenix LLC, Chandler, AZ, USA) was taken every morning. It is important to note that the protein dosing was equivalent to >0.25 g/kg BW per meal, which has been shown to be the optimal intake for muscle protein synthesis [3
]. Recently, it has been shown that women supplementing with whey protein and exercise training have increased lean mass compared to placebo supplements [4
CON participants followed a similar healthy meal plan as PRISE but included a normal protein intake (5–6 meals/day at 1.0 g/kg BW/day on all days), three of which were supplemented (Nature Valley Protein Chewy Bars®: 190 kcals, 10 g protein, 14 g carbohydrate, 12 g fat; Nature Valley Sweet and Salty Nut Granola Bars®: 170, 4 g protein, 20 g carbohydrate, 8 g fat—General Mills, Inc., Minneapolis, MN, USA—and Horizon Organic Milk®: 150 kcals, 8 g protein, 22 g carbohydrate, 2.5 g fat—WhiteWave Foods Company, Inc. Broomfield, CO, USA). On exercise days, they also consumed a caffeine (tea or coffee with sweetener: ~85 mg caffeine, 8 g carbohydrate) and electrolyte beverage (Gatorade G2®: 45 kcals, 12 g carbohydrates, 250 mg sodium, 75 mg potassium—PepsiCo, Purchase, NY, USA) along with a multi-vitamin/mineral (One-A-Day Multivitamins®—Bayer, Whippany, NJ, USA) taken every morning. CON participants were also asked to return empty food packets to monitor compliance. It is important to note, by study design, the only macronutrient that was intentionally different between groups was the protein per kg BW. Participants in both groups were given a 1-week supply of the supplements and asked to return empty packets before they received the next week’s supply as a means of assessing their compliance. Both groups were provided equivalent nutritional support and similar caloric intakes throughout the 12-week intervention.
The timing of meals was an important component of the current study, and both groups consumed meals using an identical meal pattern schedule. On resistance (R) and interval (I) exercise days (See below), participants consumed a small snack (~250 kcals) prior and, on stretching (S) and endurance (E) days, arrived fasted but well hydrated and were allowed to consume the electrolyte beverage as needed on all exercise days. Breakfast was consumed after the exercise, and remaining meals were consumed at 3-h intervals throughout the remainder of the day. On non-exercise days, participants consumed breakfast within an hour of waking in the morning and remaining meals at 3-h intervals thereafter (Table S2
2.4. RISE Exercise Training Protocol
Subjects in both groups underwent the same closely supervised/monitored progressive multiple exercise training regimen as described previously [2
]. Briefly, the training program consisted of four specific types of exercise training: (1) resistance exercise; (2) interval sprints; (3) stretching/yoga/Pilates; and (4) endurance exercise (RISE training) (Table S1
). Subjects underwent four exercise sessions per week, and the sessions rotated through the four types of exercise such that each of the four exercises was performed 1 day per week. To familiarize participants with the individual exercises and to ensure compliance, all training sessions were performed in the Skidmore College Sports Center under the close supervision of the research team. Intensity level was monitored at every exercise session with heart rate monitors (Polar H7, Polar Electro, Lake Success, NY, USA) to ensure subject safety and proper compliance with the exercise program.
Specific details of the four types of exercises that comprise the RISE training have been previously published [2
] and are shown in Table S1
. Briefly, the resistance (R) training sessions were completed within 60 min and consisted of a dynamic warm-up, footwork and agility, lower and upper body resistance, and core exercises, all performed at a resistance to induce muscular fatigue in 10–15 repetitions and for 2–3 sets. A 30-s recovery was provided between sets, and a 60-s recovery was allowed between different exercises. The sprint interval (I) training sessions were completed within 35 min and consisted of either 7 sets of 30 s “all-out” with a 4-min recovery or 10 sets of 60 s “almost all-out” with 2 min of rest after each interval. Participants were allowed to perform the sprints using any mode of exercise. The stretching (S) routine incorporated traditional yoga poses with additional stretches and Pilates movements, providing a total body stretching, flexibility, and strengthening workout. All sessions were completed within 60 min and were led by a certified yoga instructor. Finally, endurance (E) exercise training was performed for 60 min at a moderate pace (60% of maximal effort). Participants were allowed to choose from a variety of aerobic activities, including running, cycling, rowing, swimming, etc
2.6. Cardiometabolic Biomarkers
Blood lipids and C-reactive protein: A 12-h fasted venous blood sample (~20 mL) was obtained (Week 0 and Week 13). Blood was collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 2500 rpm at 4 °C. Upon separation, plasma was stored at −70 °C in aliquots until analyzed. Plasma C-reactive protein and insulin concentrations were determined using commercially available ELISA kits (Millipore, Billerica, MA, USA). Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TRGs) were assessed using the Cholestech LDX blood analysis system (Hayward, CA, USA). The test–retest intraclass correlation (r) and coefficient of variation (CV) in our laboratory with n = 15 are as follows: TC and HDL-C (mg/dL); r = 0.95, CV = 3.2%; r = 0.97, CV = 5.3%, respectively.
Heart rate and blood pressure:
Resting heart rate and systolic and diastolic blood pressure (BP) were obtained in the supine position as previously described [2
]. Heart rate and BP were obtained following a minimum of 10 min of quiet resting.
Vascular health was assessed using pulse contour analysis (augmentation index) and pulse wave velocity (Arteriograph, version 18.104.22.168, TensioMed Kft., Budapest, Hungary). Augmentation index was determined by the following formula:
is the early (direct) wave’s amplitude; P2
is the late (reflected) systolic wave’s amplitude; and PP equals the pulse pressure.
The aortic pulse wave velocity (PWVao) was determined by the wave reflection generated from the early direct pulse wave as it is reflected back from the aortic bifurcation. Return time (RT) is determined by measuring the time interval between peaks from the early direct (P1
) and reflected late (P2
) systolic waves. The PWVao calculations were measured using the distance from the upper edge of the pubic bone to the sternal notch (Jugulum-Symphisis¼), as this provides the closest approximation of the actual aortic length. PWVao was calculated with the following formula:
where RT is return time; and Jug–Sy is the aortic distance (Jugulum–Symphisis). The test–retest intraclass correlation (r) coefficient of variation (CV) in our laboratory with n
= 10 are as follows: PWV and RT; r
= 0.94, CV = 11.2%; r
= 0.90, CV = 12.0%, respectively.
2.10. Physical Performance Assessments
Following a familiarization session for all testing procedures, physical performance outcomes were assessed at Weeks 0 and 13 at the same time of day and completed over a 2-day period. For example, aerobic power (5-km TT), muscular endurance (sit-ups/push-ups), flexibility (sit and reach), and balance (standing stork balance) were completed on Day 1, whereas upper and lower body strength (bench press/leg press) and power tests (squat jumps/bench throws) and vertical jumps were completed on Day 2 (See below).
Upper Body Muscular Endurance. Upper body muscular endurance was assessed with timed push-ups in 1 min. Women started in the plank position balancing on the knees with arms extended and hands placed under the shoulders. A successful push-up was defined as lowering the body so that elbows reached 90° followed by a return to the starting plank position. Participants were asked to perform as many push-ups as possible within 60 s in a continuous pattern with no more than two seconds of rest between repetitions.
Core Muscular Endurance. Timed sit-ups were performed in the supine position with arms folded across the chest, knees bent at 90°, and feet flat on the ground and supported by a research team member. A successful sit-up required participants to curl up to a 90° position (vertical) to the floor and then return to the starting position. The sit-up action was continuous, with a rest duration of no more than 2 s allowed between repetitions. Participants were instructed to perform as many sit-ups as possible in 60 s.
Standing Balance. Postural balance was assessed with the stork balance test. While in the standing position, participants were instructed to balance on the dominant leg with the heel lifted off the ground and the non-dominant knee flexed to 90°, with the foot placed gently against the inside of the dominant knee. Hands were placed on the hips at the level of the iliac crests. The trial ended when the heel of the dominant leg touched the floor, the hands came off of the hips, or the non-dominant foot was removed from the dominant standing leg. Participants were provided three attempts and the best time was recorded for analysis.
Flexibility. Lower back and hamstring flexibility were assessed with the sit-and-reach test. This was administered using a standard sit-and-reach box (Lafayette Instrument Company, Lafayette, IN, USA), following a standard technique. The maximal distance reached of 3 trials was recorded.
5-km Cycle Ergometer Time Trial. Subjects arrived to the laboratory for performance testing sessions having consumed a standardized meal (PRISE, IsaLean bar; CON, granola bar) 1 h prior. Before the time trial began, seat and handle bar lengths, height, and tilt were adjusted according to each subject’s preferences. Each adjustment was recorded and used for the post-test (Week 13). Following a 5–7 min warm-up at 60% of heart rate reserve (HRR) on the Velotron Dynafit Pro cycle ergometer (Racermate, CompuTrainer 3D Software, Version 1, Seattle, WA, USA), participants completed a 5-km time trial (5-km TT) as fast as possible. Pedaling cadence and gear ratio were selected freely by the participant during each ride (Weeks 0 and 13). Subjects were permitted to drink water, if needed (ad libitum). Total time to complete the time trial and mean and max watts were all recorded. HR and blood pressure were recorded every five minutes during the time trial immediately upon finishing and 5 and 10 min after completion.
Upper and Lower Body Maximal Strength
. Measures of one repetition maximal strength (1 RM) of the upper and lower body were assessed via the bench (barbell) and leg press, respectively, as previously described [7
]. The test–retest intraclass correlation (r) and coefficient of variation (CV) in n
= 15 are as follows: chest 1-RM and leg 1-RM; r
= 0.99, CV = 1.6%; r
=0.99, CV = 2.7%, respectively.
Upper and Lower Body Maximal Force and Power. Following 1 RM’s of the bench and leg press, dynamic maximal force and power of the upper and lower body were assessed with bench throws (BTs) and jump squats (JS’s), respectively, using the Ballistic Measurement System (Innervations Inc., Muncie, IN, USA) interfaced with a commercial smith rack. Prior to performing the tests participants were provided instructions on how to perform the tests safely and with proper techniques. During the familiarization process, subjects performed 3–5 un-weighted practice trials for the BTs and JS’s. For the JS’s, participants performed three consecutive repetitions with the barbell loaded to 30% of their predetermined IRM for the leg press. Participants began the JS’s in the standing position with feet slightly wider than hip width apart and the loaded barbell across the upper trapezius muscles. When instructed, they lowered into the squat position until 90° of knee flexion was achieved, then jumped as high as possible, and landed with bent knees. Immediately upon landing, without pause, participants repeated the same upward jumping movement for a total of three maximal JS’s in succession.
For the bench throws (BTs), participants followed identical familiarization procedures as the JS’s by performing 3–5 un-weighted practice trials lying supine on a bench with hands positioned on the barbell slightly wider than shoulder width apart and arms fully extended. The bar was then loaded with 20% of the 1 RM of the bench press. To initiate the BTs, subjects lowered the barbell to the chest just above the distal end of the sternum and were instructed to explosively push and then release the barbell with the intent to project the barbell as high as possible. Participants caught the bar on its descent and immediately, without pause, initiated another maximal BT until 3 successive repetitions were completed. Throughout both the JS and BT tests, spotters were present on both sides of the barbell to provide verbal encouragement and ensure safety of the participants. The physical performance variables measured and used for analysis were mean and peak power (watts) taken as an average of the three repetitions.