Single Effort in Air Temperature of Minus 10°C

2.1 Study group characteristics The study involved 30 healthy males, students of the University of Physical Culture in Krakow, Poland, faculty of physiotherapy. Their mean body height was 178.9 ± 6.5 cm, mean body mass equaled 76.8 ± 11.3 kg. After the subjects were assigned to each group, the average body height in the experimental group (177.9 ± 5.9 cm) and in the control/neutral group (179.9 ± 7.2 cm) was calculated. The average body mass in the experimental group (73.1 ± 6.6 kg) was lower than that in the control group (81.2 ± 14.1 kg).

Simple randomization with a 1:1 allocation ratio served to randomly assign the participants to the experimental or control group. This was done before the first blood sampling, with the randomization sequence generated prior to the study. No stratification factors were used. The participants in the experimental group performed physical exertion under the conditions of low temperature (-10°C). The workout was undertaken in a thermal climate chamber in the Laboratory for Climate Technology Research and Heavy Duty Machines, Cracow University of Technology in Krakow. The thermal climate chamber meets the requirements of the PN-EN ISO/IEC 17025:2005 standard and possesses an accreditation certificate No. AB 1678, issued by the Polish Centre for Accreditation. The Laboratory for Climate Technology Research and Heavy Duty Machines is a research center accredited in the field of defense and security by the Minister of National Defense (accreditation No.: 55/MON/2018). The control group, in turn, performed the same physical effort but in the open air, in thermoneutral conditions (the air temperature was 18.1°C on the day of the study).

The experiment comprised 3 stages: (1) Before the exercise, blood samples were taken from the participants. This was performed in the morning hours on 11 May 2024 at the Blood Physiology Laboratory of the Central Research and Development Laboratory, University of Physical Culture in Krakow. (2) Both groups undertook an exertion test. (3) Blood samples were collected from all participants again after the exercise.

The study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Regional Medical Chamber in Krakow, Poland 2.2 Research method A two-factor experimental design with repeated measures was employed in the study. Ambient temperature (low vs. thermoneutral) constituted the between-subject factor; time of measurement (pre- and post-exercise) was the within-subject factor. The study followed the design of an exploratory investigation. In the group allocation of the participants, the principle of double randomization was employed. In experimental research, where a phenomenon is induced by the researcher on purpose, its course is observed in a planned and controlled way. Therefore, the influence of particular factors on the studied parameters can be assessed. Conclusions are drawn on the basis of the discovered relationships; the accuracy of the conclusions is evaluated with a specific probability. In the present study, a significance level of α = 0.05 was assumed, corresponding to a 95% confidence level.

2.3 Parallel group technique The participants were randomly allocated to the experimental group and the control group; the double randomization principle was followed. The first measurement preceded introducing the experimental factor.

The two groups were observed but only the experimental group received the additional stimulus, namely low temperature, i.e. the experimental factor. A second measurement was then performed, allowing to evaluate the changes imposed by the intervention. Owing to the application of a control group, it was possible to state if the reported changes in erythrocyte deformability resulted from the experimental factor - and not from other variables, which were uncontrolled. Although confounding variables are not regarded as essential in the main analysis, they can impact on the research results, leading to effect misinterpretation. Here, exposure to low ambient temperature constituted the experimental factor. The testing environments were different in the two groups (controlled thermal climate chamber in the experimental group and natural outdoor conditions in the control group), which was taken into consideration when interpreting the results.

2.4 Shuttle run test In the current study, cardiorespiratory capacity was determined with the use of an endurance shuttle run test. This is a standardized, incremental protocol commonly applied to provoke progressively increasing cardiorespiratory load. Direct physiological intensity indicators were not recorded but the same protocol was used in both groups in order to obtain comparable external workload progression.

The test started with a steady pace, which equaled 8 km/h. Subsequently, the running speed increased by 0.5 km/h per minute. The subjects ran a 20-m distance back and forth and then touched the finish line with one foot. The running pace was indicated by a beep. The participant finalized the test if they failed to reach the line at a distance of at least 3 m before the beep sound for two successive running sections. The number of completed sections was considered the test result.

2.5 Methods of blood analysis A qualified nurse collected fasting blood samples in 10-ml Vacuette tubes in the morning hours. The staff at the Blood Physiology Laboratory of the Central Research and Development Laboratory, University of Physical Culture in Krakow supervised the procedure, following the applicable standards. Serum indicators were determined in the laboratory of Diagnostyka S.A. in Krakow.

2.6 Morphological assessments The morphological analyses of blood included red blood cell count (RBC) [106/mm3] and hemoglobin (HGB) [g/dl] and were performed with the use of an ABX Micros 60 analyzer (Horiba ABX SAS, Montpellier, France).

2.7 Red blood cell aggregation index A Laser-Assisted Optical Rotational Red Cell Analyzer (Lorrca) (MaxSis Lorrca®, RR Mechatronics, Zwaag, The Netherlands) was applied. The aggregation parameters of aggregation index (AI), amplitude of aggregation (AMP), and half-time kinetics of total aggregation (T½) were evaluated with the Lorrca MaxSis Osmoscan (MaxSis Lorrca®, RR Mechatronics, Zwaag, The Netherlands).

2.8 Biochemical parameters Testosterone concentration [nmol/l] was evaluated with an Alinity I, Abbott immunochemical analyzer. The level of cortisol (μg/dl) was established with chemiluminescence (Abbott, Abbott Park, IL, USA). To determine lactate concentration, the enzymatic method (ARCHITECT c System, Abbott, USA) was used.