Effects of Hyperbaric Oxygenation on Oxidative Stress and Cool Down in Athletes: Standard Pressure Versus Low Pressure


Subjects will be recruited through public announcements in local gyms and gathered to explain
the protocol. Those willing to participate will sign a written informed consent and
recruited. To be included, all the subjects will undergo a general medical screening to allow
hyperbaric treatments. This will include weight, height, non-invasive arterial blood
pressure, and heart rate measurements.

After inclusion, subjects will be randomly assigned to three arms using an electronic number
generator by personnel not directly involved in the experiment:

– Arm 1(control): no intervention.

– Arm 2 (L-HBO): treated with oxygen at 1.45 ATA for 60 min (inclusive of compression and
decompression times, and an air break of 3 minutes breathing air);

– Arm 3 (HBO): treated with oxygen at 2.5 ATA for 60 min (inclusive of compression and
decompression times, and an air break of 3 minutes breathing air).

Subjects included in Arm 2 and 3 will undergo a total of 20 treatments. They will follow a
personalized diet proportional to their energetic expenditure.

The Authors will identify 5 time-points in the protocol:

TIME 0 (T0): immediately after inclusion, before any treatment or experiment; TIME 1 (T1):
standardized physical exercise 1, before HBO treatments; TIME 2 (T2): after HBO treatments ;
TIME 3 (T3): standardized physical exercise 2, after the end of HBO treatments; TIME 4 (T4):
2 months after the end of HBO treatments.

The following exams will be performed on the included subjects:

– at T1 and T3, subjects will undergo a standardized physical stress test on a treadmill.
Subjects will start with a fast walking at 4 km/h for 5 minutes, then will start running
at 8 km/h for 3 minutes, and then speed will increase 2 km/h every 3 minutes until VO2
max or exhaustion is reached. Lactate production will be measured on capillary blood
through finger stick at basal levels (before exercise), at the maximal threshold (VO2
max or exhaustion), then 5, 10, 15, 20, and 30 minutes after stopping the exercise while
walking at 4 km/h (dynamic cool down).

– a standardized panel including Complete Blood Count (CBC), creatinine, Blood Urea
Nitrogen (BUN), C reactive protein, and VES will be performed at T0, T2, and T4.

– oxidative stress markers will be analyzed on blood and urine samples, taken at several
steps during the experimental period. On blood samples (T0; T1; T3), the Authors will
measure IL-1 beta, IL-6, TNF-alfa, reactive oxygen species and total antioxidant
capacity (by paramagnetic resonance), nitrite and nitrate (NO2/NO3) plasma concentration
(by colorimetry based on the Griess reaction), inducible Nitric Oxide Synthase (by ELISA
commercially available kit), total (tot) and reduced (red) aminothiols (by fluorescence
spectroscopy). On urine samples (T0; T2; T4), the Authors will assess lipid peroxidation
by measuring 8-isoprostane concentration (by competitive immunoassay).

Blood samples (approximately 6 ml) will be drawn from the veins of the forearms
(preferentially on the non-dominant limb); plasma and erythrocytes will be separated by
centrifuge at 1000×g for 10 min at 4°C. Urine samples will be collected by voluntary voiding
in sterile containers. All samples will be stored in multiple aliquots at − 80 °C until
assayed and thawed only once before analysis.

With this setting, blinding of patients and investigators will be impossible due to different
structural characteristics. However, outcome assessors will be blinded to patients’


Oxidative Stress



Start Date:

March 3, 2020


University of Padova

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