The primary objective of this study was to analyze the effects of different caffeine dosages on resting and post-exercise cardiac autonomic modulation. While higher cardiac parasympathetic and global modulations were observed after CAF3 and CAF6 ingestion during the resting condition, no such effects were noted for the PLA and CON groups. Further, while all treatment groups demonstrated a significant reduction in lnRMSSD and lnSDNN 5-min following exercise, no between treatment effects were noted. Finally, given the continual HRV suppression for the PLA and CON groups at 35-min post-exercise, compared to the restoration of said indices for the CAF3 and CAF6 treatments to levels not significantly different from baseline, we conclude that the CAF ingestion in the quantities used in this study are sufficient to accelerates post-exercise autonomic recovery following a single bout of strenuous exercise. Based on these observations we accept our research hypothesis.
The effects of caffeine ingestion on resting HRV are conflicting, with studies reporting increases (30-32), reduction (33) and no changes (34) of resting parasympathetic and/or global modulation markers. Establishing a cause of these divergences is not an easy task since several variables can affect HRV analysis, such as sex (35), body position (36), body mass index (37), nutritional status (38), functional condition (39), corresponding heart rate (40), cardiorespiratory fitness (41) and age (42). In that same sense, the physiological and functional response to caffeine ingestion also depends on various factors such as individual caffeine habituation (43), caffeine dosage (44), sex (45), functional condition adopted to analysis (34), genetic profile (46), caffeine expectancies (47) and some other neuromuscular characteristics (48). Thus, it is plausible to infer that the autonomic response to caffeine ingestion is dependent on several independent variables, and the increase of cardiac parasympathetic and global modulations observed in this study may be limited to our study design and participants' characteristics.
Regarding the caffeine dosage effect, no differences between CAF3 and CAF6 on resting autonomic dynamics were observed by this study. Previous studies showed that both 2 (30) and 5 mg∙kg-1 (32) of caffeine, dosage close to those adopted in this study, were able to increase cardiac parasympathetic modulation. In this scenario, our results reinforce the possibility of increasing parasympathetic modulation after caffeine intake and add important information suggesting that the relationship between caffeine dosage and parasympathetic reactivity is not linear. No changes on lnRMSSD and lnSDNN were observed after 45 min of resting on PLA and CON groups, suggesting no effect of resting time on rest cardiac autonomic modulation. In opposition, a significant effect of resting time on supine and orthostatic cardiac parasympathetic and global modulation was previously observed after 60 minutes of resting in the supine position in young men (34). In this same scenario, Zimmermann-Viehoff et al. (2015) observed a significant effect of resting time on HRV parameters in a sample of young men and women from 30 to 50 minutes of rest at the seated position (49) . Despite ours and Zimmermann-Viehoff et al (2015) studies using the seated position to analyze HRV, some differences between them and may explain the conflicting results observed. These differences include the amplitude of R-R interval segments used for HRV analysis, rest time before the nutritional intervention and sample characteristics. Thus, these data indicate that the effect of resting time on HRV may be protocol-dependent and should be considered in studies involving the effect of different pharmacological and non-pharmacological interventions on HRV.
In the initial post-exercise analysis, all treatment groups demonstrated a significant reduction in lnRMSSD and lnSDNN, but no differences between treatments were noted. However, after 35 min of passive recovery no differences between rest and post-exercise lnRMSSD and lnSDNN were identified in CAF3 and CAF6 protocols, while a persistent depression of these autonomic markers was identified in CON and PLA groups. Thus, these results confirm our initial hypothesis (please, check it) that caffeine intake can boosts post-exercise cardiac autonomic recovery. Corroborating our results, Rolim et al. (2018) observed a higher post-exercise cardiac parasympathetic reactivation after a submaximal exercise test in young men after caffeine uptake (3 mg∙kg-1), despite no changes in resting markers of cardiac autonomic modulation (34). On the other hand, Kliszczewicz et al. (2018) underwent ten physically active young males to Wingate anaerobic test, and no effect of a complex containing caffeine (100 mg) + Citrus Aurantium (100 mg) was observed on post-exercise parasympathetic and sympathetic activity, despite higher resting sympathetic activity compared to the placebo condition (17). Otherwise, Bunsawat et al (2014) suggest that caffeine can promote a sympathetic over activation after maximal exercise, a hypothesis based mainly on higher absolute heart rate and blood pressure after an exercise test (14). However, higher training load and maximum heart rate were observed in Bunsawat’s study after caffeine intake, with no differences in heart rate recovery at the first and the second-minute post-exercise, which makes the caffeine-induced sympathetic over activation hypothesis questionable. In other words, higher post-exercise absolute heart rate and blood pressure may occur due to a higher training load and not necessarily a direct effect of caffeine ingestion.
From a physiological perspective, the autonomic response to caffeine uptake is complex and may be bilateral. Was previously reported that caffeine ingestion could promote a significant increase in plasma levels of catecholamines (50, 51) and inhibits the enzymatic degradation of cyclic adenosine monophosphate by phosphodiesterases, which potentiates postsynaptic neurotransmission in the sympathetic nervous system (52) On the other hand, despite parasympathetic response to caffeine uptake remain underexplored, it has been shown that caffeine can stimulate acetylcholine receptors and acts as an inhibitor of acetylcholinesterase (53, 54), which explains, at least in part, the caffeine-induced increase in parasympathetic activity reported in our and previous studies. In addition, it has been hypothesized that the caffeine-induced parasympathetic activation may be a result of baroreflex activation due to an increase in peripheral vascular resistance and blood pressure resulting from antagonistic caffeine effect on adenosine receptors (34), which need to be confirmed in future studies.
Notwithstanding a lack of difference between group mean power observed in caffeine and placebo protocols, higher peak power was observed in CAF3 and CAF6 compared to PLA and control reveal an ergogenic effect of caffeine on anaerobic performance. Also, higher peak power in CAF6 compared to CAF3 indicate that this ergogenic effect is dose dependent. Previously, a lack of effect and even reduction in anaerobic performance after caffeine consumption has already been reported in the literature (50). However, a recent meta-analysis using studies of good and excellent methodological quality reveal that caffeine intake can augment mean and peak power output on the Wingate anaerobic test by 3% and 4%, respectively (11) . Interestingly, in our study, higher cardiac parasympathetic reactivation after caffeine intake was observed even in the face of higher peak power in CAF3 and CAF6 compared to control and PLA protocols. This finding strengthens the favorable effect of caffeine on post-exercise parasympathetic reactivation since an inverse relationship between exercise intensity and the magnitude of parasympathetic reactivation is expected (55, 56).
While a higher fatigue index was found following caffeine compared to controlthere were no differences compared to placebo. Of note, examining the effect of caffeine supplementation on repeated bouts of Wingate tests (four 30-s Wingate tests with 4 min of rest between each exercise) after caffeine (6 mg∙kg-1) or placebo ingestion, Greer, McLean, and Graham (1998) observed that caffeine ingestion had an ergolytic effect in the latter two exercise bouts (50). Otherwise, it was recently reported that caffeine supplementation (6 mg∙kg-1) increased the peak power during Wingate anaerobic test and diminished neuromuscular fatigue, shown by attenuation of decrease in countermovement jump performance after Wingate test (57) . Thus, since increase (57, 58) and reduction (50, 59) of different markers of exercise tolerance after caffeine supplementation already been reported, the recommendation of caffeine supplementation to improve recreational or athletic performance should be made cautiously.
Despite no observed difference between RPE in caffeine and placebo during warm-up, the main effect of treatment and lower RPE observed after CAF6 compared to control and PLA indicates that caffeine may reduce the exercise-induced psychological stress. Interestingly, Duncan et al (2019) observed a reduction of RPE during Wingate test for the upper-body, but not for the lower-body segment, suggesting that caffeine’s effect on RPE depends on body segment exercised (60). Despite the absence of caffeine effect on RPE during lower-body Wingate test observed in some studies (60-62), our findings reveal that this benefit can be acquired with caffeine supplementation in this condition. We note that lower RPE identified in CAF6 protocol was accompanied by high peak power and mean power, which reinforce the psychostimulant effect of caffeine. It is an interesting approach since increases in exercise performance without altering RPE mean a higher power output without the increase in psychological stress per se; this positive effect should also be investigated in future studies.
As expected, an increase of BLA was observed after WAnT in all protocols indicating the vital contribution of anaerobic metabolism to the energy requirements during the exercise test. Despite increase (63) and maintenance (60) of BLA levels are commonly reported after caffeine intake, lower BLA concentration was observed in CAF3 compared to other protocols after five minutes of recovery. Unfortunately, the only lactate analysis performed in the initial phase of recovery does not permit to detect the exact moment with the highest lactate concentration, which makes any inference about the effect of caffeine on lactate production or clearance questionable. In the final phase of post-exercise recovery, we observed higher BLA levels in CAF6 compared to PLA, but the absence of difference between CAF6 and control prevents the attribution of higher blood lactate to caffeine supplementation. Of note, blood lactate reflects the balance between lactate production and clearance and the precise mechanisms that explain the small differences observed in this study is unclear and it may be just a inter day variation of BLA response to exercise, hypothesis previously reported in the literature (64, 65).
Limitations: A major strength of our study is our randomized, crossover design. A limitation of our study is the use of a single, acute bout of WAnT testing. Therefore, we cannot generalize our findings to higher exercise volume conditions, such as multiple WAnT testing, multiple sets of resistance training, interval style workouts. We also cannot generalize our findings to women. The absence of ventilatory, sympathetic activity, and post-exercise blood pressure analysis, variables that influence HRV could also contribute to a better physiological interpretation of our data. We believe that a particular strength of our study was the use of a non-supplemented CON condition in addition an inert PLA and support this contention that a number of between group comparisons in our study were significant vs. the CON, but not the PLA treatments. Lastly, we assessed cardiac parasympathetic reactivation during 35 minutes of recovery, which limits our conclusions to this time window. However, despite the mentioned limitations, the analysis of autonomic response to different caffeine supplementation dosages on resting and post-exercise conditions adopted in this study adds robust information to current scientific debate about the autonomic effect of caffeine ingestion. The post-exercise time window adopted in this study allow fast and slow parasympathetic reactivation analysis and is within of window of opportunity for sudden death in young observed 30 minutes after vigorous exercise, which can be partially attributed to post-exercise cardiac autonomic dysfunction (66) and add clinical relevance to our results.