## 2 Materials and Methods
### Study Variables  
Clinical variables were gender, age, height, weight, BMI and knee pain intensity, assessing using a 1-10 cm VAS (0 = no pain, 10 = worst pain ever), with mild, moderate and severe pain categorized as scores >4, between 4 and 6 and >6, respectively. VAS scores were measured at visits 1 and 2, as well as every morning on getting out of bed and in reference to the intensity of pain experienced on the previous day. Daily VAS scores were recorded in a personalized notebook for each patient and were then analyzed at weekly intervals. The Western Ontario McMaster Universities Arthritis Index (WOMAC) was used to assess pain, stiffness and function in patients with OA. The subscales of pain, stiffness and function have 5, 2 and 17 items, respectively. The test was completed by the volunteer subjects taking into account pain, stiffness and knee functionality in the past 48 h. The test questions are scored on a scale of 0-4 (from 0 = none to 4 = extreme). Higher WOMAC scores are indicative of worse pain, stiffness and functional limitations. A Spanish validated version of the WOMAC index was administered [48]. Body composition was analyzed by bioelectrical impedance analysis (BIA) on a whole-body BIA analyzer: Tanita BC-420 MA (Tanita Corporation, Tokyo, Japan). Variables analyzed included weight (kg), fat mass (kg), percentage of fat mass and muscle mass (kg). The subject's mobility and balance was evaluated using the Timed Up and Go test (TUG). Briefly, subjects are seated properly in a chair with their arms resting (not on the armrest) and are instructed to stand up, walk 3 m, make a turn around the chair and sit down. The recorded time on the stopwatch is the subject's TUG score. Two tests were per-formed, with a resting time of 30 s. The average recorded time of the two tests was the final score. A TUG score \(\geq\) 13.5 s is predictive of a risk of falls, whereas a score < 13.5 s suggests better functional performance [49]. The muscle function of the leg with knee pain was measured by isokinetic and isometric dynamometry. These tests were carried out with the Biodex System 3 isokinetic dynamometer (Biodox Medical Systems, Shirley, New York, NY, USA). The measurement protocol included a 5-min warm-up on a cycloergometer (W45, 70-75 rpm). The dynamometry was performed in a seated position with 90\({}^{\circ}\) hip flexion and the knee module aligned with the axis of the knee movement. The pad that exerted resistance during movement was adjusted to the central part of the tibia. The range of motion was set to 0\({}^{\circ}\)-90\({}^{\circ}\), taking the maximal active knee extension as the initial value. Subjects were instructed to perform 5 repetitions of maximal knee flexion and knee extension (concentric/concentric) at 60\({}^{\circ}\) and 180\({}^{\circ}\)-s\({}^{-1}\). Warm-up included 2 sets of repetitions. The variables measured were the isokinetic value of relative peak torque (expressed in Newton meters [Nm]/body mass [Kg]), total work (TW) measured in joules (J), average power measured in watts (W) and total work per repetition maximum (total word for 1 RM) measured in J. In isometric dynamometry (at 90\({}^{\circ}\)), the force exerted by the subject when attempting to perform full extension of the knee was measured. The force had to be maintained for 5 s, while the lever did not allow leg movement. A total of 3 repetitionswere performed, with a rest period of 40 s between each repetition. The variables measured were maximum isometric torque (Nm) and mean maximum isometric torque (Nm). After a 12 h fast, at visits 1 and 2, peripheral blood samples were drawn for standard laboratory tests to measure biomarkers of inflammation and matrix cartilage degradation, including interleukin-1 beta (IL-1\(\beta\)), matrix metalloproteinase-3 (MMP-3) or stromolysin-1 and cartilage oligomeric matrix protein (COMP). Plasma levels of IL-1\(\beta\) were measured using a commercial kit (Human IL-1 beta ELISA Kit, ref: E-EL-H0149, Elabscience Biotechnology Inc., Houston, TX, USA), with results expressed as pg/mL.