Methods
illness or injury, PBC
BLOOD, HEART, PBC
The study was approved by both the Australian Institute of Sport Ethics Committee (approval number 20121211) and the University of Sydney Ethics Committee (approval number 2013/0430). Testing was conducted at the New South Wales Institute of Sport in Sydney in a randomised study design.Male participants aged between 18 and 35 years were recruited by advertisement at the University of Sydney. Inclusion criteria included completion of the Physical Activity Readiness questionnaire (PAR-Q) and General Health Questionnaire (GHQ), and a screening for any recent illness or injury, or for the current use of antioxidant supplements, or anti-inflammatory or steroidal medication which may have impacted immune competence.In total, 55 individuals were screened, 15 were excluded due to illness or injury, inability to commit to the trial requirements, or current use of anti-inflammatory medication. The 40 remaining participants provided informed consent and were randomly allocated a treatment (PBC Participants were asked to refrain from physical activity and alcohol the day before testing and to abstain from caffeine and food intake 2 h prior to the testing session. Blood samples were taken prior to treatment exposure, and again, immediately after. At each of the collection time points, 9 mL of blood was collected into a heparin-coated vacutainer (BD Biosciences, San Jose, CA). Cell concentrations in the blood sample were determined using a Sysmex XP-300™ Automated Hematology Analyzer (Sysmex, Kobe, Japan).HR and blood pressure were measured at the end of 15 min of seated rest and immediately after exposure. Heart rate (HR) was recorded using a wrist HR monitor (RS800CX Training Computer, Polar Electro, Kempele, Finland) and chest strap (H3 Heart Rate Sensor, Polar Electro, Kempele, Finland). Blood pressure was measured using an automatic self-inflatable cuff (HEM-7121 Automatic Blood Pressure Monitor, Omron, Kyoto, Japan).During PBC treatment, participants stood upright with the head exposed in a single user cryocabin wearing gloves, soft boots, and shorts (Space Cabin Cryosauna Classic, Kherson, Ukraine) for 3 min between − 110 and − 140 °C following a safety protocol that has been referenced elsewhere (Fonda and Sarabon PBMCs were isolated from 9 mL of whole blood collected in heparin tubes according to previously published methods (Jaatinen and Laine PBMC populationsObservations of cell populations at 15 min of rest, and immediately post-exposure. ± Standard deviation, *denotes significant time effect of treatment group between rest and post-exposure, Results from the rest and post time points between the three treatments were analysed using a two-way repeated-measures ANOVA (treatment × time) using SPSS software (IBM SPSS Statistics 24 Software, New York, USA), with a 95% confidence interval. All data are presented as mean ± standard deviation (SD). The Shapiro–Wilk test of normality was used to determine the distribution of data. Levene’s test of equality of error variances was used to verify the assumption that variances were equal between groups. The variance of means between groups at rest was analysed from results of the one-way repeated-measures ANOVA to ensure that there were no statistically significant differences in resting concentrations of variables between groups prior to treatment. The Bonferonni adjustment was used to reduce the risk of Type I error, and pairwise comparisons were used in cases that a significant effect was reported. Raw data were used, with outliers and missing data included in group analysis but not in any pairwise comparisons, and are reflected in the degrees of freedom reported.