*4.2. Experimental Procedure*

All horses were trained 6 days per week and the training program consisted of 7 runs of 1000 m each at di fferent speed. Briefly, the first warm-up run clockwise at light trot (mean speed 5 m/s) was followed by 3 counter clockwise runs (mean speed 10 m/s), then 1 recovery run at walking and 2 runs at full speed trot (mean speed 12 m/min), and finally 2 runs at light trot (mean speed 5 m/s). All animals were sampled the day before the rest-day.

Saliva and blood samples were collected before (T0) and immediately after full speed exercise (T1). Saliva samples were collected by using cotton swabs and Salivette ® tubes (Sarstedt AG & Co., Nümbrecht, Germany). The cotton swab was grasped with a surgical clamp, inserted at the angle of the lips into the mouth of the horse and placed gently on the tongue surface for 5 min and then inserted into the Salivette ® tube.

Venous blood samples were collected at T0 and T1 by jugular venepuncture into 4 mL vacutainer sterile tubes containing EDTA (Vacuette ® Greiner Bio-One, Cassina de Pecchi, Italy) and 9 mL vacutainer sterile tubes containing clot activators (Vacuette ® Greiner Bio-One, Cassina de Pecchi, Italy). A complete blood count (CBC) and biochemical profile was performed on blood and serum samples to exclude systemic disorders.

Whole blood was directly tested in the field for blood lactate concentration by using a portable device (Accutrend Plus ®, Roche, Mannheim, Germany) at T0 and T1 as a part of the fitness evaluation routine.

All the sampling procedures were performed before feeding, at the same time of the day (06:00–8:00 a.m.) and, immediately after collection, all the samples were stored at 5 ◦C and delivered to the lab within 2 h.

## *4.3. Laboratory Analysis.*

Saliva samples were obtained by centrifugation of Salivette® tubes (10 min at 1000 g) (Universal 32, Hettich Zentrifugen, Tuttlingen, Germany) and 2 mL aliquots of supernatant were stored at −20 ◦C until metabolomic analysis.

Blood samples with clot activators were centrifuged for 10 min at 1000 g (Universal 32, Hettich Zentrifugen, Tuttlingen, Germany) and the obtained sera were stored at −20 ◦C until analysis.

For 1H-NMR analysis, we created a stock solution composed of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mmol/L and NaN3 2 mmol/L in D2O. The former served as the NMR spectra chemical-shift reference, while the latter avoided bacteria proliferation. The solution was set to pH at 7.00 ± 0.02 by phosphate buffer (1 M). Both serum and saliva samples were prepared for 1H-NMR by thawing and centrifuging 1 mL of each sample at 4 ◦C for 15 min at 18,630 g. The supernatant (700 μL) was added to 100 μL of the NMR analysis solution and centrifuged again.

The spectra were recorded with an AVANCE III spectrometer (Bruker, Milan, Italy), controlled by the Topspin software (Ver. 3.5), at a frequency of 600.13 MHz and a temperature of 298 K. The residual signal from the water was suppressed by pre-saturation, while broad signals from large molecules were reduced by a CPMG-filter, set as outlined by Zhu et al. [58]. Each spectrum was acquired by summing up 256 transients registering 32 K data points over a 7184 Hz spectral window, with an acquisition time of 2.28 s and relaxation delay of 5 s.

In Topspin, a manual correction phase was applied to each spectrum, together with a line-broadening of 0.3 Hz. The subsequent steps were performed in R computational language by means of scripts developed in-house. The spectra were aligned toward the right peak of the alanine doublet, set to 1.473 ppm. Baseline was then corrected, after having removed the residual water signal, by isolating irregularities of the baseline by peak detection, according to the "rolling ball" principle [59].

For signals' assignment, chemical shift and multiplicity were compared with the Chenomx software library (Chenomx Inc., Edmonton, AB, Canada, ver 8.3). The added TSP was employed as internal standard in the first sample analyzed. Differences in water content among samples were then taken into consideration by probabilistic quotient normalization [27]. Rectangular integration was employed to quantify each molecule, by focusing on one signal per molecule free from superimpositions.
