2.3.4. Statistical Procedures

Statistical analyses were carried out using the software Statistica (version 13.1; Statsoft, Inc., Tulsa, OK, USA). For all analyses, significance was accepted at *p* < 0.05. Descriptive statistics are represented as mean ± standard deviation (SD) with standard mean difference data. Tests of normal distribution and homogeneity (Kolmogorov–Smirnov and Levene's, respectively) were conducted on all data before analysis. Paired sample *t*-test was used for determining differences as a repeated measures analysis (pre–post). Cohen d was the effect size indicator. To interpret the magnitude of the effect size, we adopted the following criteria: d = 0.20, small; d = 0.50, medium; and d = 0.80, large [37]. A Pearson's correlation coefficient r was used to examine the relationship between the percentage of change of all biological mark [100 − (post × 100)/pre) and the training load (urine, sleep quality, stress, fatigue, soreness, Hooper Index, RPE general, RPE breath, RPE neuromuscular, sRPE

general, sRPE breath, and sRPE neuromuscular [100 − (post × 100)/pre]). To interpret the magnitude of these correlations, we adopted the following criteria [37]: r ≤ 0.1, trivial; 0.1 < r ≤ 0.3, small; 0.3 < r ≤ 0.5, moderate; 0.5 < r ≤ 0.7, large; 0.7 < r ≤ 0.9, very large; and r > 0.9, almost perfect. Regression analysis was used to model the prediction of SMD blood biomarkers from remaining variables with positive correlation.

#### **3. Results**

First, a paired measure *t*-test with hematological parameters (WBC, RBC, RCDW, Hb, MCV, MCHb, MCHbC, MPLTV, EOS%, BASO%, NEUT%, LYMP%, MNC%, ALC, and AEC) showed no significant differences between before and after the pre-season period. There was a significant increase in PLT, while a significant decrease in AMC and ANC after the pre-season period (see Table 1, for more information).

**Table 1.** Before and after pre-season data (mean ± SD) of anthropometric and hematological parameters (HP).


HP: Hematological parameters; WBC: White blood cells; RBC: Red blood cells; Hb: Hemoglobin; Ht: Hematocrits; MCV: Mean corpuscular volume; MCHb: Mean corpuscular hemoglobin; MCHbC: Mean corpuscular hemoglobin concentration; RCDW: Red cell distribution width; PLT: Platelets; MPLTV: Mean platelets volume; NEUT: Neutrophils; LYMP: Lymphocytes; MNC: Monocytes; EOS: Eosinophils; BASO: Basophils; ANC: Absolute neutrophils count; ALC: Absolute lymphocytes count; AMC: Absolute monocytes count; AEC: Absolute eosinophils count; \* Denotes significance at *p* < 0.05, and \*\* denotes significance at *p* < 0.01.

> A new paired measures *t*-test with biochemical parameters including, potassium, albumin, ferritin level, TC, TG, C-HDL, and, C-LDL, showed no significant differences between before and after the pre-season period. There was a significant increase in creatinine, ALP, CRP, cortisol, and testosterone, while a significant decrease in calcium and calcium corrected after the pre-season period (see Table 2, for more information).


BcP: Biochemical parameters; ALP: Alkaline phosphatase; CRP: C-reactive protein; TC: Total cholesterol; TG: Triglycerides; C-HDL: High-density lipoprotein cholesterol; C-LDL: Low-density lipoprotein cholesterol; \* Denotes significance at *p* < 0.05, and \*\* denotes significance at *p* < 0.01.

> At this point, testosterone/cortisol ratio (T/C ratio) was calculated. In fact, the T/C ratio has been considered as an important physiologicaal variable to gauge individual condition and responses. In this sense, a *t*-test with data form the T/C ratio showed the same values before (0.317 ± 0.05) and after (0.324 ± 0.04) pre-season period [*t*(25) = 2.13, *p* = 0.07, d = 0]. Testosterone/cortisol ratio over the period can be found in Figure 1.

**Figure 1.** Before and after pre-season data (mean ± SD) of testosterone/cortisol ratio.

Regarding training load data, a paired measures *t*-test revealed no significant differences between before and after the pre-season period for urine color, stress, fatigue, sleep quality, and soreness measures. There was a significant increase in the Hooper Index, RPE (general), RPE (breath), RPE (neuromuscular), sRPE (general), sRPE (breath), and sRPE (neuromuscular) after pre-season compared to before pre-season (see Table 3, for more information).


**Table 3.** Before and after pre-season data of training loads (mean ± SD).

RPE: Rate of perceived exertion; sRPE: Session rate of perceived exertion; A.U.: Arbitrary units \* Denotes significance at *p* < 0.05, and \*\* denotes significance at *p* < 0.01.

> Table 4 shows the relationships between percentage change of training load and the percentage of changes in hematological parameters. Very large positive correlations between RPE (general) and MCN% (*r* = 0.73; *p* = 0.001), and very large negative correlations between RPE (neuromuscular) and NEUT% (*r* = −0.71; *p* = 0.002) were found. Large positive correlations were found for the Hooper Index (*r* = 0.67; *p* = 0.004), soreness (*r* = 0.61; *p* = 0.01), and fatigue (*r* = 0.57; *p* = 0.02) with ALC percentage of changes. In addition, large positive correlations between sRPE (general) (*r* = 0.60; *p* = 0.012), RPE (neuromuscular) (*r* = 0.53; *p* = 0.03), and MNC percentage of changes were found. While, moderate negative correlations were found between stress and EOS (*r* = −0.50; *p* = 0.04) percentage of changes.

> The associations between the percentage of change of training load and the percentage of changes of biochemical parameters can be seen in Table 5. Large negative correlations between sRPE (general) and sodium (*r* = −0.60; *p* = 0.013), between sleep quality (*r* = −0.58; *p* = 0.01), stress (*r* = −0.53; *p* = 0.033) and albumin, and between urine (*r* = −0.51; *p* = 0.04) and creatinine percentage of changes were found. On the other hand, large positive correlations between sRPE (breath) (*r* = 0.60; *p* = 0.014) and testosterone, and between RPE (general) (*r* = 0.56; *p* = 0.02) and C-HDL percentage of changes were found. While, moderate positive correlations between RPE (neuromuscular) (*r* = 0.50; *p* = 0.04) and ALP percentage of changes were found.



HP: MCHbC: Mean corpuscular hemoglobin

MNC: Monocytes; EOS: Eosinophils; BASO: Basophils; ANC: Absolute neutrophils count; ALC: Absolute lymphocytes

count; \* Denotes significance at *p* < 0.05.

Hematological

 parameters; WBC: White blood cells; RBC: Red blood cells; Hb: Hemoglobin;

concentration;

 RCDW: Red cell distribution width; PLT: Platelets; MPLTV: Mean platelets volume; NEUT: Neutrophils;

 Ht: Hematocrits;

 MCV: Mean corpuscular volume; MCHb: Mean corpuscular hemoglobin;

 count; AMC: Absolute monocytes count; AEC: Absolute eosinophils

 LYMP:

Lymphocytes;



Low-density

 lipoprotein cholesterol; \* Denotes significance at *p* < 0.05.

A multilinear regression analysis was performed to verify which variable of percentage of change of training load (agreement with the correlation analysis) could be used to better explain the percentage of change of hematological and/or biochemical parameters.

The percentage of change of urine color was a predictor of the percentage of change of creatine (*r* = −0.51). The percentage of change of sleep quality was a predictor of the percentage of change of albumin (*r* = 0.58). The percentage of change of stress was a predictor of the percentage of change of EOS and albumin (*r* = −0.50 and *r* = −0.53). The percentage of change of fatigue was a predictor of the percentage of change of ALC (*r* = −0.57). The percentage of change of soreness and hooper index were predictors of the percentage of change of ALC (*r* = 0.61 and *r* = 0.67), respectively. The percentage of change of RPE (general) was a predictor of the percentage of change of MNC and C-HDL (*r* = 0.73 and *r* = 0.56), respectively. The percentage of change of RPE (neuromuscular) was a predictor of the percentage of change of NEUT, MNC and ALP (*r* = −0.71, *r* = 0.53, and *r* = 0.50), respectively. The percentage of change of sRPE (general) was a predictor of the percentage of change of MNC and sodium (*r* = 0.60 and *r* = −0.60), respectively. The percentage of change of sRPE (breath) was a predictor variable of the percentage of change of testosterone (*r* = 0.60) (see Table 6. for more information).



\* Denotes significance at *p* < 0.05.
