The goal of this study was to quantify the effect of a traditional Italian conditioning method (CM). Six Standardbred yearlings were kept in standardized conditions for 140 days. From T1 (day 1) to T10 (day 10), the horses were hitched for 30 minutes/day to accustom them to a cart. From T11 to T70, they were jogged or exercised for 30 minutes/day at a slow trot (4.4 ± 0.3 m/second) and from T71 to T140 for 40 minutes/day (5 m/second ± 0.3 m/second). Every 15 days, the horses performed fast exercise for a distance of 5,000 m with increased speeds. Blood samples were collected before and after daily training (DT) every 21 days (P1…P7), to measure the following variables: erythrocytes (RBC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), total protein (Pt), albumin, aspartate amino transferase (AST), alkaline phosphatase (ALP), creatine kinase (CK), lactate dehydrogenase (LDH), urea, creatinine, glucose, triglycerides, and blood lactate. A three-step standardized exercise test (SET) also was performed. Analysis of variance was used to study differences during the training. Tukey's post-hoc test was used for statistical multiple comparison (P ≤ 0.05 and 0.01). Lactate level did not show an increase over 4 mmol/L in all periods. Glucose was higher at P1 than P7. There was no correlation between method of conditioning and the level of CK, LDH, or AST. Conditioning method induced an increase in triglycerides for the change in exercise metabolism supply. According to SET, conditioning method induced an increase of SVO2max (speed [m/second] reached to a theoretical maximal oxygen consumption), V4 (speed [m/second] reached at a lactate content of 4 mmol/L), V200 (speed [m/second] at 200 beats/minute) and HR4 (heart rate [beats/minute] at 4 mmol/L of lactate). We concluded that the Italian conditioning method used in this study improved the fitness of horses.