**2. Materials and Methods**

The article reported according to STROBE (the Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for cohort designs [31].

#### *2.1. Study Design and Setting*

The present study followed an observational analytic cohort design with a quasiexperimental (pre-post) design. The period of data collection occurred between 2 June (beginning of the pre-season) and 19 September (after pre-season) of 2019. On 2 June and 19 September, players were assessed for their biological markers. Between the periods, the players were daily assessed for the training load parameters and wellbeing. From the blood samples collected to measure the biological markers, hematological and biochemical parameters were analyzed. All players were internally monitored in all training sessions during the pre-season period. All internal loads were monitored using subjective measures. For the quantification of subjective internal loads, the rate of perceived exertion (RPE) and the session-rate of perceived exertion (s-RPE) for general, breath, and neuromuscular perceived exertions were applied.

#### *2.2. Participants and Study Size*

Twenty-five professional soccer players (mean ± SD; age 28.1 ± 4.6 years old, height 176.7 ± 4.9 cm, body mass 72.0 ± 7.8 kg, and body fat percentage 10.3 ± 3.8%; body mass index using Quetelet equation: 23.4 kg/m2), from a professional club competing in the first league of Qatar (2019/2020 season), participated in this study. The inclusion criteria were (i) completed blood samples collections before and after pre-season period; (ii) no history of any neuropsychological impairments that could affect the results of the experiment (iii) absence of injuries, physical constraints, or illnesses during study period; (iv) absence of fatigue or illness during the blood samples collections of before and after the pre-season period; (v) participating in a minimum of 80% training sessions during the study period; and (vi) not have taken drugs such as pain killers or others that may influence the biochemical status during the two weeks before assessments. Technical staff and professional soccer players were informed regarding the study design and its related benefits and risks, as well as the main aims of the current investigation. All players signed an informed consent form to voluntarily participate in this study. All the professional soccer players in this study were treated according to the American Psychological Association (APA) guidelines, which ensure the anonymity of participants' responses. The study protocol was approved by the Scientific Committee of School of Sport and Leisure (Melgaço, Portugal) with the code number CTC-ESDL-CE00118. The study followed the ethical standards of the Declaration of Helsinki.

#### *2.3. Variables, Data Sources, and Quantitative Variables*

#### 2.3.1. Anthropometry

Anthropometric measures were performed before and after the pre-season period, at the same time of the day. Body mass was measured using a body composition monitor (HD-351, Tanita, Arlington Heights, IL, USA) to the nearest 0.1 kg. While, the height was measured using a stadiometer to the nearest 0.1 cm (Seca 217, Ham- burg, Germany). Fat mass was also estimated using the body composition monitor. All measurements were performed by the same professional with a level 2 certification from the International Society for the Advancement of Kinanthropometry (ISAK). The experienced professional was considered mainly for the case of ensuring accuracy and precision in anthropometric measures related to height. Moreover, this professional also ensured the reproducibility conditions for the case of body composition analysis using bioimpedance. Those conditions were related to the protocol of cleaning the machine every time a player was measured, waiting the same time between players and after cleaning, and ensuring the same player's position during the measurement.

#### 2.3.2. Biological Markers

#### Hematological Parameters

Laboratory blood samples were collected from players' antecubital vein in a seated position. Blood samples (15 mL) were collected between 8:00 and 10:00 am, before and after the pre-season period. The blood samples were collected with all players in fasting, and with at least 12 h of rest (the time between the last training session, and the second blood draw) before the laboratory blood tests. All blood samples were centrifuged at 2500 rpm for 10 min, and the serum of each sample was immediately frozen at −80 ◦C for later biochemical analysis. Furthermore, 3 mL of blood were collected into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA). The blood samples were analyzed through flow cytometry, using a flow cytometer (FACSCaliburTM, BD Biosciences, San Jose, CA, USA) and using an automated hematology analyzer (Sysmex kx-21N Kobe, JAPAN). This method allowed to obtain hematological variables as follow: WBC: White blood cells; RBC: Red blood cells; Hb: Hemoglobin; Ht: Hematocrit; MCV: Mean corpuscular volume; MCHb: Mean corpuscular hemoglobin; MCHbC: Mean corpuscular hemoglobin concentration; RCDW: Red cells distribution width; PLT: Platelets; MPLTV: Mean platelets volume; NEUT: Neutrophils; LYMP: Lymphocytes; MNC: Monocytes; EOS: Eosinophils; BSO: Basophils; ANC: Absolute neutrophils count; AMC: Absolute monocytes count; ALC: Absolute lymphocytes count; and AEC: Absolute eosinophils count.
