Nitrate Formulations and Exercise Performance

this study compares the acute effects of Nitrate and Nitrate plus citrulline supplementation on physical performance in recreationally trained males and females. This is a cross-over within-participant comparison design concluding three arms (i) 600 mg of Nitrate (N), ii) 600 mg Nitrate and Citrulline (NC) and (iii) Placebo (PL).

Conditions N, NC and PL will be tested one week apart, considering a minimum washout period of 72 hours.

Dependent variables:

* Number of completed repetitions to failure in squat using 75% of 1RM.
* Ventilatory thresholds (VT1) and (VT2) and Vo2 max from a progressive to exhaustion running test (PGT)
* Lactate post (within 1 min of having completed the PGT)
* Rating of perceived exertion (RPE) by the Borg 6-to-20-point scale recorded every 2 min and at the end of the PGT.
* Heart rate associated with VT1, VT2, and Vo2max - Heart rate maximum.
* Maximal aerobic speed (MAS): calculated from the average speed of the last 2 minutes of the PGT.
* time limit test at MAS (conducted after 15 min of having completed the PGT)

Aim:

To analyse and compare the acute effect of two different nitrate formulations on strength and endurance performance in young recreationally trained individuals. In a cross-over within-participant comparison design the participants will be assigned either to ingest (i) 600 mg of Nitrate (N), ii) 600 mg Nitrate and Citrulline (NC) and (iii) Placebo (PL).

Design: Cross-over randomised controlled within participant design including one group undertaking three different treatment conditions.

1. Nitrate (N) providing 600 mg (9.6 mmol) of Nitrate (NO-3).
2. Nitrate + Citrulline (NC) providing 600 mg (9.6 mmol) of Nitrate (NO-3) and 6 g of citrulline-malate.
3. Placebo (PLA) Participants (15 or higher) Inclusion criteria: (a) males and females 18-40 years of age; (b) physically active participants with experience in resistance training performing squat and endurance running as habitual exercises (c) free from musculoskeletal limitations or injuries, (d) agree not to ingest any other supplement during the study.

Exclusion criteria: (a) a history of various metabolic conditions or diseases; (b) use of a variety of medications, including but not limited to those with androgenic and/or anabolic effects and/or nutritional supplements known to affect training outcomes such as creatine, proteins, etc. within 6 weeks before the beginning of the study, (c) current use of tobacco products.

All participants must provide written informed consent by the Declaration of Helsinki. The University ethics committee must approve procedures before starting with the data collection.

Procedures Day 0: Familiarization with the testing procedures including the progressive test to determine the 1RM value in Squat.

Diet Record: Each participant will complete a 1-week diet record using a food frequency questionnaire (FFQ). This questionnaire consists of questions relating to the weekly consumption of different types of food, to estimate energy consumption and the nutritional composition of the reported diets. Participants were instructed to maintain their habitual diet throughout the study. If any change in diet patterns is identified, the participants were dropped from the study.

Supplementation protocol:

Days 1 to 3 Supplements (N, NC, or PLA) will be ingested 2 hr before a standard low-volume training session including 3 sets of 8 to 10 reps of squat and deadlift using a very light load (e.g., own body mass), and flexibility exercises following by 5 min of running at self-paced intensity rated as light (RPE 12). This routine can be completed at home.

Day 4: supplements will be ingested 2.5 hours before the assessments.

The three conditions Nitrate (N) Nitrate + Citrulline (NC) and Placebo (PL) will be tested one week apart, considering a minimum washout period of 72 hours, a strategy that has previously been used in studies with a similar design.

All participants will be following an identical training week in each of the three treatment conditions.

Dependent variables:

o Number of completed repetitions in a singular set to failure in a squat with 75% of 1RM.

Lactate Post (within 1 min of having completed the squat) 15 min rest Progressive to exhaustion on a treadmill (PGT)

* Ventilatory thresholds (VT1) and (VT2)
* Vo2 max
* Lactate post (within 1 min of having completed the PGT)
* Rating of perceived exertion (RPE) using the Borg 6-to-20-point scale recorded every 2 min and at the end of the PGT.
* Heart rate associated with VT1, VT2, and Vo2max - Heart rate maximum.
* Maximal aerobic speed (MAS): calculated from the average speed of the last 2 minutes of the PGT.

  15 min rest
* Maximal Aerobic Speed test (no cortex).
* Lactate pre (15 min post PGT)
* The participants should run until exhaustion at the previously determined MAS (e.g. 19.8 km/h)
* RPE after 1 minute and at the end
* Heart rate every 1 minute and at the end
* Lactate post (within 1 minute of having completed the test

Eligibility Criteria:
Inclusion Criteria

* Physically active participants with experience in resistance training performing squats and endurance running as habitual exercises
* Free from musculoskeletal limitations or injuries
* Agree not to ingest any other supplement during the study.

Exclusion Criteria

* History of various metabolic conditions or diseases
* Use of a variety of medications, including but not limited to those with androgenic or anabolic effects or nutritional supplements known to affect training outcomes such as creatine, proteins, etc. within 6 weeks before the beginning of the study
* Current use of tobacco products.