Cocoa-flavanols enhance moderate-intensity pulmonary V̇O2 kinetics but not exercise tolerance in sedentary middle-aged adults: A randomised controlled trial


 Background: Cocoa flavanols (CF) may exert health benefits through their potent vasodilatory effects which are perpetuated by elevations in nitric oxide (NO) bioavailability. These vasodilatory effects may contribute to improved delivery of blood and oxygen to exercising muscle.Objective: Therefore, the objective of this study was to examine how CF supplementation impacts pulmonary oxygen uptake (V̇O2) kinetics and exercise tolerance in sedentary middle-aged adults.Methods: We employed a double-blind cross-over, placebo-controlled design whereby 17 participants (11 male, 6 female; mean±SD, 45±6 years) randomly received either 7 days of daily CF (400 mg) or placebo (PL) supplementation. On day 7, participants completed a series of ‘step’ moderate- and severe-intensity exercise tests for the determination of oxygen uptake kinetics.Results: During moderate-intensity exercise, the time constant of the fundamental phase of V̇O2 kinetics (τV̇O2) was decreased by 15% in CF as compared to PL (mean±SD; PL: 40±12 vs. CF: 34±9 s, P=0.019), with no differences in the amplitude of V̇O2 (AV̇O2; PL: 0.77±0.32 vs. CF: 0.79±0.34 l min−1, P=0.263). However, during severe-intensity exercise, τV̇O2,the amplitude of the slow component (SCV̇O2) and exercise tolerance (PL: 435±58 vs. CF: 424±47 s, P=0.480) were unchanged between conditions.Conclusions: Our data show that acute CF supplementation enhanced oxygen uptake kinetics during moderate-, but not severe-intensity exercise in middle-aged participants. These novel effects of CFs, in this demographic, may contribute to improved tolerance of moderate-activity physical activities, which appear commonly present in daily life.Registered under ClinicalTrials.gov Identifier no. NCT04370353

TDV O2, time delay of the fundamental response; Tlim, limit of exercise tolerance; V O2, oxygen 23 uptake; V O2b, baseline oxygen uptake; τV O2, time constant of the fundamental response. Skeletal muscle contraction and force production form the basis for the ability to perform 51 physical activity, both for daily life activities as well as during sports-related events. Repeated 52 muscle contractions require continuous regeneration of adenosine triphosphate (ATP). The 53 production of ATP during (prolonged) physical activity is driven through oxidative 54 phosphorylation, which depends on sufficient delivery of oxygen (O2) (1). Impairment of 55 pathways involved in the delivery of O2 to working skeletal muscle, such as with older age or 56 physical inactivity, leads to slower rates of pulmonary O2 uptake (V O2) and therefore greater 57 O2 deficit (2-4). Impaired V O2 kinetics in response to physical activity are associated with 58 reduced exercise tolerance (5-7) and may also affect capacity to perform daily life activities 59 that require moderate-intensity physical activity. 60 61 The slower dynamic adjustment of V O2 observed in older adults across a metabolic transient 62 is thought to be due to a mismatch of O2 delivery to O2 utilisation. Indeed, attenuations in 63 microvascular blood flow supply and distribution (and thus O2 delivery) within aged skeletal 64 8 On the 7th day of supplementation, participants were advised to consume 4 capsules 45 min 151 prior to arrival at the laboratory. The supplementation protocol was chosen so that participants 152 commenced exercise testing ~90 min following supplement ingestion, which coincided with 153 reported peak plasma flavanol concentrations (27). The participants completed a series of 154 "step" exercise tests from an unloaded (0 W) baseline to moderate and severe-intensity work 155 rates for the determination of pulmonary V O2 kinetics. Tests began with 3 minutes of 0 W 156 baseline cycling, before a step change in power output to 80% GET or 60% for 6 minutes and 157 until Tlim, respectively. Participants completed three bouts of moderate-and one bout of severe-158 intensity exercise, each separated by 10 min of passive recovery. This protocol was employed 159 with the knowledge that prior moderate-intensity exercise does not impact the V O2 kinetics of 160 subsequent heavy intensity exercise (28,29). 161

Measurements 163
During all exercise tests, pulmonary gas exchange and ventilation were measured at the mouth 164 breath-by-breath using a metabolic cart (Jaeger Oxycon Pro, Hoechberg, Germany). 165 Participants wore a facemask and breathed through a low dead space (90 ml), low resistance 166 (0.75 mmHg l−1 s−1 at 15 l/s) impeller turbine assembly (Jaeger Triple V, Hoechberg, 167 Germany). The inspired and expired gas volumes and gas concentration signals were 168 continuously sampled at 100 Hz, the latter using paramagnetic (O2) and infrared (CO2) 169 analysers (Jaeger Oxycon Pro, Hoechberg, Germany) via a capillary line connected to the 170 mouthpiece. These analysers were calibrated before each test with gases of known were time aligned by accounting for the delay in capillary gas transit and analyser rise time 174 relative to the volume signal. Breath-by-breath fluctuations in lung gas stores were corrected 175 for by computer algorithms (30). Heart rate was measured during all tests via short-range 176 radiotelemetry (Polar H10,Polar Electro,Kempele,Finland). During one of the transitions to 177 moderate-and severe-intensity exercise for both supplementation periods, a blood sample was 178 collected from a fingertip over the last 30 s preceding the step transition in work rate and within 179 the last 15 s of exercise. Blood samples were immediately analysed using a hand-held device 180 (Lactate Pro, Nova Biomedical, USA) to determine blood lactate concentration. Blood lactate 181 accumulation was calculated as the difference between blood lactate at end exercise and blood 182 lactate at baseline. 183 184 After arrival to the laboratory, participants underwent an assessment of the previous 7 days 185 physical activity levels and sedentary behaviour by the International Physical Activity 186 Questionnaire (IPAQ) and by accelerometery (ActiGraph GTX3). Following 10 min of seated 187 rest, participants blood pressure was measured in the brachial artery. Blood pressure was 188 measured three times and the mean of the responses was recorded. 189

Data analysis 191
Breath-by-breath V O2 data were edited to remove data points lying more than 3 standard 192 deviations (SD) outside the local 5-breath mean (31). The resultant data were then linearly 193 interpolated to provide second-by-second values. For V O2 and heart rate data in response to 194 moderate exercise transitions, second-by-second data for the three transitions were averaged 195 together to produce a single dataset. The severe-intensity exercise bout for each condition was 196 not repeated and was modelled separately. For each exercise transition, the first 20 s of data after the onset of exercise (i.e., the cardiodynamic or phase I response) were deleted (32,33) 198 and a mono-exponential model (Equation 1) with time delay was then fitted to the data (4), as 199 follows: 200 Peak V O2 was 2.45±0.61 l min−1 (28.1±5.7 ml kg−1 min−1), with the mean GET occurring at 240 1.51±0.46 l min−1 (108±39 W). The peak work rate attained from the incremental test was 241 207±49 W and the work rates calculated to require 80% of the GET and 60% were 87±29 W 242 and 166±40 W, respectively. 243 The V O2 kinetic parameters for moderate intensity exercise are presented in Table 2, and the 267 V O2 response of a representative participant to moderate-intensity exercise is shown in Figure  268 3. Compared with PL, τV O2 was smaller during moderate-intensity exercise following CF The pulmonary V O2 response to severe-intensity exercise for a representative participant is 274 shown in Figure 4A and group mean responses are shown in Figure 4B. The associated 275 modelled parameters are presented in Table 2. No impact of CF supplementation on the τV O2 276 (P=0.799) for exercise initiated at 60% ∆ over PL was evident. There were no differences in 277 V O2b (P=0.246), AV O2 (P=0.427), TDV O2 (P=0.617), SCV O2 (P=0.887), Gain (P=0.640), or 278 end exercise V O2 (P=0.954) between conditions. TD SCV̇O 2 was lower following CF vs. PL and Tlim (P=0.480) were not significantly different following PL and CF supplementation 281 during severe-intensity exercise (see Table 2  The purpose of this study was to examine the impact of CFs on pulmonary V O2 kinetics during 312 two intensities of cycling exercise in healthy, normotensive middle-aged individuals. 313 Congruent with our hypothesis, the major finding of this study was that 7-days CF 314 supplementation enhanced pulmonary V O2 kinetics during moderate-intensity exercise as 315 demonstrated by a significant reduction in τV O2. These effects of CFs, however, were not 316 apparent during severe-intensity exercise when compared with a PL. Ultimately, the findings 317 of the present study may have clinical potential in contributing to improved tolerance of daily 318 life activity in middle-aged adults. 319 320

Effects of CFs on the Physiological Responses to Moderate-Intensity Exercise 321
This study is the first to investigate whether CFs modulate pulmonary V O2 kinetics. We show 322 that 7 days CF supplementation significantly reduced the τV O2 (40 vs. 34 s) associated with 323 the transition from unloaded to moderate-intensity cycling in middle-aged adults. Notably, the 324 magnitude of change in τV O2 (~6 s) reported is important, as it exceeds the minimum 325 physiologically relevant change of ~5 s (32). The reduction in τV O2 observed after CF 326 supplementation in our middle-aged individuals reflects a shift towards oxygen kinetics 327 typically observed in younger healthy individuals (35), whereby V O2 kinetics are not limited 328 by O2 delivery per se (36). Theoretically, a lowered τV O2 would reduce the O2 deficit incurred 329 during the exercise transition, thereby causing less perturbations to the intracellular milieu (i.e., 330 ∆ phosphocreatine, ADP, H+, inorganic phosphate, glycogen) and enhancing exercise tolerance 331 (5-7). Therefore, our data suggest CFs may lower the O2 deficit incurred during moderate-332 intensity activity by negating age-associated impairments to O2 delivery and pulmonary V O2 333 kinetics. 334 Since the purpose of the study was to examine the impact of CFs on V O2 kinetics, our data 336 raise the question about the potential underlying mechanisms contributing to the lowered τV O2 337 with CF supplementation. It is acknowledged τV O2 is sensitive to manipulations in O2 delivery 338 (36,37). Further, the slowing of V O2 kinetics with advancing age occurs primarily as a 339 consequence of lowered O2 availability in oxidative skeletal muscle (2,8,10 future studies should investigate a potential muscle fibre-type dependency of CF 373 supplementation on the physiological responses to exercise. Another potential explanation for 374 the differences between exercise intensity domains presented herein relates to the dose of CFs 375 administered. Recent published evidence suggests that the 400 mg CF prescribed is the 376 minimum dose necessary to exert beneficial effects during exercise (39). Therefore, the dose 377 used in the present study may not have been high enough to raise blood flow sufficiently during 378 severe-intensity exercise to detect a measurable effect upon V O2 kinetics. In addition, CFs had 379 no beneficial impact on resting systolic or diastolic blood pressure over PL, which may be 380 attributable to insufficient dosage (55). Besides, another limitation of the study is that only a 381 single bout of severe-intensity exercise was conducted. As we were unable to feasibly include 382 additional visits for testing, we could not carry out multiple severe-intensity bouts to enhance 383 the signal-to-noise ratio of these V O2 responses and potentially detect differences between

CONCLUSION 387
In the present study, seven days supplementation with a flavanol-rich cocoa-extract resulted in 388 a reduced τV O2 following moderate-, but not severe-intensity exercise in normotensive, 389 middle-aged adults. Whilst the O2 cost of exercise was similar between CF and PL, the faster 390 V O2 kinetics response at the onset of moderate-intensity exercise suggests an improvement in 391 O2 delivery with acute CF intake. Such effects were not found with severe-intensity exercise. 392 Overall, CF administration may reduce the metabolic perturbations associated with moderate-393 intensity exercise in middle-aged adults.    Pulmonary V O2 responses of a representative participant displayed with associated τV O2. 600 Panel B) Group mean V O2 responses during the rest-to-exercise transition following PL and 601 CF supplementation. Group mean ± SD V O2 at limit of exercise tolerance also shown.