NO^• has a vital role in regulating vasodilation, blood flow, and muscle oxygenation; thus, increasing NO^• bioavailability positively affects performance during exercise and recovery [13,42,43]. Consequently, supplementation with NO^• precursors such as L-citrulline and L-arginine to augment nitric oxide bioavailability and enhance performance is a common practice followed by athletes and physically active individuals. Of interest, L-citrulline supplementation appears to be more efficient in enhancing NO^• bioavailability compared to L-arginine because it bypasses hepatic metabolism, increasing this way the levels of extracellular L-arginine [3,5,17]. Indeed, previous studies reported that chronic L-citrulline supplementation might increase nitric oxide bioavailability [5,44]. In the present study, we found that a single dose of 6 g of L-citrulline increased exhaled NO^• one hour after the supplementation, which is supported by relevant studies that showed that after oral L-citrulline supplementation, L-arginine concentrations peak around one hour later [45,46] in a dose-dependent manner [5]. Thus, based on our results, a single dose of 6 g of L-citrulline supplementation 1 hour before exercise is sufficient for increasing NO^• bioavailability.

Certainly, considering the different origins of nitric oxide in skeletal muscle (i.e., neuronal, and endothelial) and exhaled air (i.e., epithelial) [47], some plasma or skeletal muscle measurements of nitric oxide production and/or metabolism could have added insightful mechanistic information. In the present study, our purpose was to examine whether acute L-citrulline supplementation would affect sternocleidomastoid muscle blood flow, oxygenation, and respiratory performance. Thus, the lack of any NO^• measurements in skeletal muscle is a significant limitation of the present study. Yet, we have particularly focused on the respiratory system and pulmonary function, and have chosen, therefore, nitric oxide in exhaled air. Additionally, blood pressure and heart rate after L-citrulline supplementation were not measured since changes in these parameters were observed after chronic L-citrulline supplementation [48]. Moreover, alterations in blood pressure after L-citrulline supplementation were observed mainly in participants with health issues. Such as obese postmenopausal women [49,50], elderly individuals [32], and heart failure patients [51], while in the present investigation, young, healthy individuals were recruited.

Inspiratory muscle dysfunction is a primary contributor to ventilatory failure during fatiguing conditions such as exercise [52]. During intense exercise, inspiratory dysfunction can occur due to increased work of breathing and/or insufficient blood flow and oxygen delivery to the respiratory muscles, progressively leading to fatigue and impairing exercise performance [25]. The sternocleidomastoid muscle is a crucial accessory muscle [53] that is highly active during exercise, supporting the primary inspiratory muscles’ work [54]. Additionally, sternocleidomastoid muscle deoxygenation has been reported to be progressively increased during incremental inspiratory loading [55]. Thus, it was hypothesized that enhancing sternocleidomastoid muscle blood flow and oxygen delivery through L-citrulline supplementation might favorably affect respiratory muscle performance and resistance to fatigue during incremental resistive breathing.

However, contrary to our hypothesis, acute L-citrulline supplementation and the concomitant NO^• increase did not improve sternocleidomastoid muscle performance and resistance to fatigue either one hour post supplementation or post-respiratory muscles resistive breathing to task failure. Comparable results have been reported in studies of the same nature using clinical and healthy populations [56,57]. However, in these investigations, the supplementation was L-arginine. L-arginine can be metabolized in the liver, contrary to L-citrulline, which mainly contributes to NO^• production [3,17]. Specifically, MIP and the number of breaths at exhaustion were not different between the L-citrulline and the placebo conditions. Additionally, FEV₁, FVC, and their ratio were not affected by the L-citrulline supplementation. Even though our resistive breathing to task failure protocol induced fatigue, perhaps it was insufficient to cause extensive disturbances on respiratory performance, as observed in other studies using resistive breathing [58,59,60]. Given the fact that sub-maximal constant endurance exercise induces significant fatigue in respiratory muscles [61], it could be suggested that future studies examining inspiratory muscle fatigue should employ constant whole-body high-intensity endurance exercise protocols to compromise blood flow to inspiratory muscles and induce fatigue. Indeed, during whole-body exercise, the manipulation of inspiratory muscle work with resistors will increase the competitiveness between inspiratory and locomotor muscles for blood flow and oxygenation [23,62]. Thus, we believe that it is more likely to find any favorable effect of a supplement on fatigue when the exercise protocol involves whole-body exercise and greater fatigue levels occur in the muscle under examination.

In the present study, we used NIRS that continuously monitors regional tissue oxygenation in vivo. We found that after both resistive breathing conditions (i.e., L-citrulline or placebo supplementation) and at every intensity stage (i.e., 70%, 80%, and 90% of MIP), there were a significant decrease in Δ[O₂Hb] and TSI% and a significant increase in Δ[HHb] compared to baseline in the sternocleidomastoid muscle, which is in line with previous studies [55]. The muscle’s oxygenation and deoxygenation responses during loading were as expected in order to facilitate oxygen supply to the working muscles. However, despite the changes we observed after incremental inspiratory muscles resistive breathing to task failure in these parameters, L-citrulline supplementation did not affect sternocleidomastoid muscle oxygenation. Considering the role that NO^• has in blood flow and the increase we observed after L-citrulline supplementation in exhaled NO^•, we expected that L-citrulline supplementation would have improved sternocleidomastoid muscle oxygenation.

Similar results were reported in a study where acute L-citrulline supplementation enhances NO^• bioavailability but had no effect on blood flow in young and older adults [63]. Additionally, no effect on blood flow was observed after ingestion of a combination of 10 g of L-citrulline with whey protein in older adults [64]. On the contrary, after longer-term supplementation with concentrate watermelon juice (providing 3.4 g/day of L-citrulline for 16 days), the tissue oxygenation index of the vastus lateralis was enhanced during moderate-intensity exercise [44]. Similarly, supplementation with L-citrulline (6 g/day for 7 days) increased VO₂ uptake kinetics of the vastus lateralis during moderate and high-intensity exercise in recreationally active adults [16]. Furthermore, muscle blood flow during exercise was improved after 14 days of L-citrulline supplementation (6 g/day) in older men [31]. Therefore, a chronic supplementation intervention could be required to successfully improve inspiratory muscle oxygenation and resistance to fatigue.