*2.2. Clinical, Physical Activity, and Diet Assessment*

Sociodemographic status, lifestyle, family history of chronic diseases, self-report of non-communicable chronic diseases, and current medication use were investigated through questionnaires. Physical examination included body mass index (BMI) assessment and blood pressure levels. Dietary intake was obtained through three 24 h-recalls and assessed in the Food Processor software (ESHA Research, 2012), with subsequent energy adjustment [17]. A physical activity questionnaire validated for the Brazilian population was applied [18–20]. This questionnaire investigates the habitual physical activity (divided into physical exercise in leisure, leisure, and locomotion activities and total physical activity score) performed in the last 12 months, associated with frequency, duration, intensity. Baecke's physical activity scores do not allow to classify physical activity, however, for each one of its sixteen questions, the points vary from 0 (zero) to 5. The final score is directly proportional to physical activity and is useful to associate with health outcomes [18–20].

#### *2.3. Biochemical Measurements*

Blood was drawn after a 12-h fast, placed in EDTA tubes (1.0 mg/mL), and erythrocytes were separated from plasma by centrifugation, and both were frozen at −80 ◦C immediately after collection. Protease inhibitors (10 μg/mL of aprotinin, 10 μg/mL of benzamidine and 5 μg/mL of phenylmethylsulfonyl fluoride) and BHT (100 μg/mL) were added to preserve samples. All samples were divided into aliquots to avoid repeated defrost cycles and storage at −80 ◦C until analyses. Plasma total cholesterol, HDL-c, TG, glucose (Labtest Diagnostica SA, MG, Brazil), Apo A-I and Apo B (Wako Chemicals USA Inc., Richmond, VA, USA), and high sensitivity *C*-reactive protein (hs-CRP) (Diagnostic System Laboratories, Inc., Webster, TX, USA) were measured by commercial kits. LDL-c was calculated according to the Friedewald equation.

### *2.4. Erythrocyte Fatty Acids Analysis*

The analysis of FA from erythrocyte membranes was performed based on a previous method [21]. After plasma separation (3000× *g*, 10 min, 4 ◦C), 300 μL of erythrocytes were washed with 5 mL of phosphate-buffered saline (PBS) solution (pH 7.4) four times. The precipitate was transferred to threaded tubes, to which 1.75 mL of methanol, 50 μL of an internal standard solution containing 1 mg tridecanoic acid (C13:0)/1 mL hexane, and 100 μL of acetyl chloride were added. Thereafter, the solution was vortexed and heated in a water bath at 90 ◦C for 1 h. After that, 1.5 mL of hexane was added, and the solution was homogenized for 1 min. The samples were centrifuged at 1500× *g*, 4 ◦C for 2 min, and 800 μL of the supernatant was transferred to a different tube. This step was repeated with the addition of 750 μL of hexane. The tubes containing the collected supernatants were placed on a centrifugal concentrator at 40 ◦C for 20 min. Then the FA methyl esters were dissolved in 150 μL of hexane and transferred to a glass insert in a vial. Analyses were conducted considering the fatty acids individually, as well as the total *n*-3 (C18:3 *n*-3 + C20:3 *n*-3 + 20:5 *n*-3 + C22:5 *n*-3 + 22:6 *n*-3), total *n*-6 (C18:2 *n*-6 + C20:4 *n*-6) and Omega-3 Index (C20:5 *n*-3 + C22:6 *n*-3), the latter having been named by Harris and von Schacky [13]. To assess biological effects of fatty acids, the following ratios were calculated: C20:4 *n*-6/C20:5 *n*-3, C18:3 *n*-3/C20:5 *n*-3, C18:3 *n*-3/C22:6 *n*-3 and C18:2 *n*-6/C18:3 *n*-3.

#### *2.5. Cardiovascular Risk Assessment*

The CV risk was assessed by FRS [1,22], Reynolds Risk Score (RRS) [23,24], and the American College of Cardiology/American Heart Association 2013 Risk Score (ACC/AHA-2013) [25]. The CV risk was stratified into three categories for each score: low, moderate, and high risk. Diabetes (i.e., glucose ≥ 126 mg/dL or current hypoglycemic medication use) was considered a coronary artery disease (CAD) equivalent [26].
