This investigation was approved by the Institutional Review Board according to the principles and policies of the Declaration of Helsinki.
Twelve well-trained male CF practitioners (mean ± SD: age = 29.5 ± 4.3 years, body mass = 79.4 ± 5.5 kg, height = 174.1±6.6 cm) volunteered to participate in this study.
Following an explanation of all experimental procedures, and the associated risks and benefits of participation, each participant provided written informed consent to participate. The following inclusion criteria were established: a) More than two years of experience in CF; b) Regional, national and/or international competition level; c) one-repetition maximum (1RM) in full back squat (FBS) greater than 120 kg; d) no cardiovascular, respiratory, metabolic, neurological or orthopedic disorders that could affect cycle ergometer performance; e) no consumption of drugs or medication; f) no smoking; and g) no consumption of any other supplement at the time of the study.
The procedures applied in the study lasted three weeks during which four sessions were performed (Figure 1). In the first week, participants were required to appear at the CF sports center twice. During the first session, a researcher explained all the experimental procedures and study details to the CF athletes. In the second session, a 1RM test was performed in FBS exercise to determine loading intensity (in kg, 50% of 1RM) that should be used to compare the two experimental conditions.
Over the next two weeks, participants completed the WOD test (sessions 3 and 4) at our Exercise Physiology Laboratory to compare the two experimental conditions (BJ and PL). For this purpose, a washout period of one week separated each test session. Both sessions were always carried out under similar ambiental conditions (temperature 20°C– 23 °C, relative humidity 45%–55%). The two experimental conditions (BJ or PL) were administered using a randomized, double-blind, crossover design. In each test session, the participants began with the WOD test protocol 3 hours after ingesting the BJ or PL. The WOD test consisted of repeating the same exercise routine twice: 1st. Wall ball (WB) shots plus FBS with a 3-min rest between the two exercises. 2nd. WB shots plus FBS without rest between the two exercises. A 3-min rest was established between the two exercise routines.
Before the WOD test, blood collection to determine lactate and hormone concentrations (cortisol and testosterone pretest) were performed under resting conditions upon arrival at the laboratory and 3 hours after the BJ or PL intake. Next, arterial oxygen saturation (SpO2) and muscular fatigue were assessed (pretest). Once the WOD test was completed, SpO2 and muscular fatigue were measured again. After completing SpO2 and mechanical fatigue assessment, blood draws were performed to examine lactate and hormone concentrations (post-test), respectively. The participants performed pretest and post-test in both experimental conditions (BJ and PL) at the same time frame (± 30 min) of day.
Participants refrained from any high-intensity physical effort from 72 h and abstained from any type of physical exercise within the previous 24 h before starting the first session to the study end. Participants maintained regular training throughout the study (~ 21 days). They were allowed to perform low intensity workouts, except 24 h before the start of the test. All the participants were completely familiarized with the experimental procedures.
Diet and beetroot juice intake
The type of diet can condition energy metabolism during exercise , therefore, a nutrition professional established nutritional guidelines to ensure that all CF athletes followed a similar diet 48 hours before starting the tests which consisted of ~60% carbohydrates (5.5 g carbohydrate per kg), 25% lipids and 15% proteins. The diet was registered by the participants 48 hours before the first experimental test, and the same diet was replicated 48 hours before the second test. Compliance with the established dietary instructions was assessed by checking the participants' diaries.
Participants were required to avoid foods with a high NO3- content at least 72 hours before the study outset, and therefore, they were provided with a list of foods (arugula, lettuce, celery, parsley, spinach, turnip, leak, cabbage, endives, beetroot). Intake of caffeine (except breakfast coffee), alcohol, or other supplements was prohibited during the study to avoid any interaction with BJ. Twenty-four hours before the tests, the participants were asked to abstain from brushing their teeth or using a mouthwash and chewing gum or sweets that could contain a bactericidal ingredient such as xylitol or chlorhexidine. Oral antiseptics can alter blood NO2- concentrations after NO3- intake due to their effects on mouth bacteria . Participants were alerted of possible side effects from BJ intake: a red appearance of urine and feces and gastrointestinal symptoms.
BJ or PL intake occurred 3 hours before the start of each test. Scientific evidence indicates that a NO2- peak in blood occurs 2-3 hours after NO3- intake . An expert in nutrition and dietetics prepared the PL drink. The PL drink was prepared by dissolving 2 g of powdered BJ (~ 0.01 mmol, 0.620 mg of NO3−, Experience-Naturgreen, Murcia, Spain) in a liter of mineral water. Lemon juice was added to simulate the taste of commercial BJ. Participants drank the BJ contained in a randomized assigned bottle containing 140 ml (~ 12.8 mmol, ~ 808 mg NO3−) of BJ Beet-It-Pro Elite Shot concentrate (Beet IT; James White Drinks Ltd. , Ipswich, UK) or PL. Both drinks (BJ and PL) were supplied in an unlabeled 140 ml garnet-red plastic bottle.
One-repetition maximum full back squat test
1RM test protocol was performed as in previous studies [44, 45] according to the guidelines established by Baechle and Earle . The FBS test included a general and specific warm-up for all subjects. The 1RM test was performed with free weight and involved several attempts using increasing weights. The 1RM was defined as the last load (kg) lifted by the participant while completing a knee extension to the required position. The rest period between each attempt was 4 minutes.
After performing a general warm-up consisting of 5 minutes of low-intensity rowing and 5 minutes of joint mobility and dynamic stretching exercises, a specific warm-up was carried out consisting of 10 push-ups, 10 walkouts and 10 squat jumps. Next, the WOD test was performed to compare the effects of the two experimental conditions (BJ vs. PL). For this purpose, two characteristic exercises in CF workouts were chosen: WB and FBS.
The WOD test consisted of performing the same exercise routine twice. In the first round, WB shots for 90 seconds plus FBS for 60 seconds were completed. A 3-min passive rest (aerobic conditions) was applied between the two exercises because 3-5 min rest allows a greater number of repetitions when performing multiple sets in loads at 50% of 1RM . A 3- to 5-min rest might be safer and more reliable from a psychological and physiological perspective . Next, the same exercise routine was repeated but without recovery time. Then, the second round involved WB shots for 90 seconds plus FBS for 60 seconds without rest (anaerobic conditions) between the two exercises. The performance goal was to achieve the highest number of repetitions within a given time domain.
The WOD protocol was designed according to the guidelines established by Glassman . CrossFit workouts can be grouped into three different categories: gymnastics, metabolic conditioning, and weightlifting. The WB and FBS exercises were classified as a weightlifting routine. The principles of "constantly varied, high-intensity, functional movement” were determined as well as a wide variety of mode, exercise, metabolic pathway, rest, intensity, sets and repetitions .
Wall ball technique
A 10 kg medicine ball was used for the WB shots. The WB shots were performed at arm's length away from the wall starting from the upright position with the knees and hips fully extended with the feet hips-width apart and the toes pointed just slightly outwards, similar to doing a squat. The medicine ball had to be picked up and held at chest-height so that the elbows were tight at the sides. From this position, the butt dropped back and down to lower into a squat while the chest was kept in an upward position with the ball against the sternum. The final position was sitting back onto the heels and trying to achieve as deep a squat as possible. Then, participants immediately reversed the motion and ascended back to the up-right position and simultaneously press, or toss, the ball to a target spot about eight feet up the wall.
Full back squat technique
FBS exercise was performed with free weight. This was executed starting from the upright position with the knees and hips fully extended. Both feet were positioned flat on the floor in parallel or slightly rotated outwards at a distance of approximately shoulder-width apart. The barbell was grasped with a closed pronated grip and placed on the upper back (trapezius muscle) at the level of the acromion. Then, the participants flexed the knees and hips (eccentric action) to descend the barbell in a controlled manner until the top of the thighs reached below the horizontal plane. From this position, the participants immediately reversed motion and ascended back to the up-right position until the knees and hips were fully extended.
Participants arrived at the Exercise Physiology Laboratory between 4:00 and 7:00 PM. Blood was collected at the same time of day at rest (pre-test) 3 hours after the BJ or PL intake and immediately after intervention (post-test).
Blood was drawn from the antecubital vein into a 10 mL EDTA Vacutainer tube. Next, serum was extracted, centrifuged at 2500 x g for15 minutes, aliquoted and stored at -80ºC until later analysis. Serum free testosterone levels were determined by the enzyme immunoassay methodology according to the manufacturer's instructions using the automated Triturus EIA analyzer (Grifols-Quest, Miami, FL, USA). Serum cortisol concentrations were analyzed using chemiluminescent microparticle immunoassay by Abbott Architect immunoassay analyzer (Abbott Laboratories, Abbott Park, IL, USA) . The coefficient of variation for the between and within assay replicates was less than 10%.
Blood lactate concentrations were measured by an experienced investigator using the analyzer Lactate ProTM 2 (Arkray Factory Inc., KDK Corporation, Shiga, Japan). Clean blood samples were obtained from the index finger of the left hand as in previous studies [19, 22]. The reliability and accuracy of this device has been reported by others .
Arterial oxygen saturation
SpO2 was estimated with the Nonin WristOx2™ pulse oximeter (Model 3150, Plymouth, MN, USA). Nonin oximeters have commonly been used for monitoring SpO2 . Concretely, the WristOx2 3150 model has an accuracy of ± 2% for SpO2 measurements  and complies with the International Organization for Standardization (ISO) standards ISO 10993-1 and ISO 80601-2-61. SpO2 was tested on the index finger of the right hand. Raw data were stored on the internal memory of the WristOx2.
Lower limb fatigue was determined by a countermovement jump (CMJ) test. The CMJ test was performed using a force platform (Musclelab, Ergotest Technology AS, Langesund, Norway) before and after the WOD test following a method described previously [44, 45]. Participants carried out 3 jumps separated by a 30-sec rest, and the mean values of vertical height recorded in the 3 CMJs were used in the statistical analysis.
The Shapiro-Wilk test was used to check the normal distribution of the data, which are reported as mean and standard deviation (SD), mean and confidence intervals (95% CI) or percentage (%). To compare the effects of the two experimental conditions (BJ vs. PL), a two-way analysis of variance (ANOVA) with repeated measures was applied (experimental condition x time). Bonferroni adjustments were used to control for multiple post-hoc comparisons. The magnitude of the response to both experimental conditions was estimated by partial eta-squared (ηp2). The scale for classification of ηp2 was 0.10 = small, 0.25 = medium, 0.40 = large . Statistical power (SP) was calculated.
The formula [(post-test - pretest) / pretest] x 100 was applied to determine the percentage changes (D%) between the pretest and the post-test in the measured variables (hormones, lactate, SpO2, CMJ). The Student’s t-test for paired data was used to determine significant differences in percentage changes between the two experimental conditions. Significance was set at p < 0.05. All statistical procedures were applied using the software package SPSS version 25.0 for Mac (SPSS Inc., Chicago, IL, USA).