Ten healthy young trained male adults who majored in physical and health education participated in this study (age: 26.0 +/− 4.4 yr, height: 171.2 +/− 6.1 cm, body-mass: 70.7 +/− 8.9 kg). They habitually performed sports such as track and field, swimming, soccer and basketball over three times per week (3.1 +/− 0.9 times/ week), with moderate to high intensity over two hours per session (2.1 +/− 1.4 hour/time). Written informed consent was obtained from all subjects after a full explanation of the experimental purpose and protocol. Moreover, the experimental protocol was approved by the Kanazawa University Health & Sports Science Ethics Committee.

The experimental design was a double-blinded crossover method. Namely, subjects participated in both conditions: L-ornithine hydrochloride supplementation and placebo (indigestible dextrin aqueous solution). Due to the cross-over design, all subjects participated in both conditions with a week wash-out period between conditions. Moreover, the test condition order was counter balanced to eliminate order effect. In addition, subjects were instructed to refrain from intensive exercise for two days prior to the experiment and fast overnight before the experiment to avoid a nutritional imbalance created by eating and drinking. Subjects were also instructed not to consume beverages or food containing caffeine during the experimental period.

Subjects ingested L-ornithine hydrochloride or an indigestible dextrin aqueous solution with the same flavor (placebo) at the ratio of 0.1 g per kilogram body mass. Isomers exist in almost all amino acids, including ornithine, and are divided into levorotatory (L-) and dextrorotatory (D-) amino acids. L-ornithine corresponds to the former group of amino acids and is naturally occurring. The hydrochloride of the L-ornithine was used in this study.

The effect of L-ornithine hydrochloride ingestion was examined using the double-blinded cross-over method stated above. A placebo was required so that both subjects and testers are not biased to the effects of L-ornithine hydrochloride. A dextrin aqueous solution with the same flavor as L-ornithine hydrochloride solution is difficult to digest. Hence, the influence of the nutrients is considered to minimal.

Subjects engaged in the experimental protocol (Figure 1) with L-ornithine hydrochloride and placebo ingestion conditions at least a week after VO_2peak measurement. They conducted resistance exercise after L-ornithine hydrochloride or placebo ingestion following 60 min rest in a semi-recumbent position after entering the laboratory. Resistance exercise was designed according to previous studies [6,22]. The resistance exercise used in this study affects blood concentrations of anabolic hormones, such as catecholamine, growth hormone and insulin, and lactic acid [6,22,23]. Subjects conducted resistance exercises in the following order: chest presses, lat pull downs, leg presses, shoulder presses, leg extensions and leg curls. Three sets of each exercise were conducted and repeated 10 times using a 10 RM load determined in the first and second sets. Subjects repeated each exercise with 85% of 10 RM until exhaustion in the third set. In addition, a 90 second rest was performed with each set. Resistance exercises in both conditions were conducted with the same load and repetition number. Ergometer bicycle exercise at 50% of VO_2peak for 60 min was conducted following a 120 min rest in a semirecumbent position after resistance exercise.

Subjects were instructed not to consume beverages or food containing caffeine during the experimental period. They were also instructed to refrain from intensive exercise for two days prior to the experiment. All experimental protocols started at 8:00 am and were completed by 1:00 pm, and they fasted overnight before the experiment to avoid a nutritional imbalance. Hydration (water) during the experiment was allowed as needed. In addition, laboratory temperature was maintained at 24˚C - 26˚C.

The initial blood draw was conducted after 60 min rest in a semirecumbent position after entering the laboratory. The blood draw was conducted immediately after resistance exercise and before and after submaximal ergometer bicycle exercise (Figure 1). Each blood sample (12 ml) was collected from the antecubital vein and analyzed for growth hormone, free fatty acid, ketone bodies and ornithine. 7 ml blood was transferred into the blood sampling tube, which contains clotting and enhancing film and serum separating medium for growth hormone, free fatty acid and ketone bodies, and 5 ml of blood were transferred into 65IU heparin sodium for ornithine analysis. These processes were carried out within 30 s of the blood draw. The samples were immediately centrifuged, and the supernatants were placed in chilled containers and stored frozen at −80˚C until analysis. Serum growth hormone concentration was analyzed by radioimmunoassay using the kits from SRL Inc., Japan. Sensitivity, inter-assay and intra-assay coefficients of variation (CV) of this assay were 0.04 ng·m/L, 4.0% and 3.4%, respectively. Serum free fatty acid and ketone body concentrations were analyzed by the enzymatic method. These interassay and intraassay CV were 0.2% and 0.9% for free fatty acid and 0.6% and 0.7% for ketone bodies, respectively. The samples for ornithine determination were analyzed by high performance liquid chromatography using the an automatic amino acid analyzer (JOEL, JLC-500/V). Sensitivity, inter-assay and intra-assay coefficients of variation (CV) of this assay were 5.92 nmol/l, 4.94% and 0.00%, respectively.

Expired gases were continuously sampled in a breathby-breath method during submaximal ergometer bicycle

exercise for 60 min. Oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were measured with an automatic expired gas analytic system (AE-280S; Minato Medical Science, Japan). Respiratory exchange ratio (RER), which is calculated by VO₂ and VCO₂, was used to estimate the relative contribution of fat and carbohydrate oxidation to total energy production (% fat and carbohydrate oxidation) [24]. RER and % fat oxidation were determined without urinary nitrogen analysis because of the negligible contribution of protein to substrate oxidation during rest [25].

Two-way repeated measures analysis of variance (ingestion condition × measurement time) was used to examine the effect of L-ornithine hydrochloride ingestion. When there was a significant main or interaction effect, Tukey’s honestly significant difference was used as post-hoc analysis to examine specific mean differences. Moreover, the paired t-test was used to examine the difference between both conditions for total energy production during submaximal endurance exercise. The effect sizes were calculated in the above stated analysis (ANOVA: partial η², t-test: cohen’s d). An alpha concentration of 0.05 was used for all tests.