The Effect of a Hydroxytyrosol-Rich, Olive-Derived Phytocomplex on Aerobic Exercise and Acute Recovery

is current scienti c interest in naturally sourced phenolic compounds and their potential bene ts to health, as well as the effective role polyphenols may provide in an exercise setting. This study investigated the chronic effects of supplementation with a biodynamic and organic olive fruit water phytocomplex (OliPhenolia ® [OliP]), rich in hydroxytyrosol (HT), on submaximal and exhaustive exercise performance and respiratory markers of recovery. Twenty-nine recreationally active participants (42 2 yrs; 71.1 2.1 kg; 1.76 0.02 m) consumed 2 28 mL d 1 of OliP or a taste- and appearance-matched placebo (PL) over 16 consecutive days. Participants completed a demanding, aerobic exercise protocol at ~75% maximal oxygen uptake ( . V

hydroxytyrosol (HT), on submaximal and exhaustive exercise performance and respiratory markers of recovery. Twenty-nine recreationally active participants (42 2 yrs; 71.1 2.1 kg; 1.76 0.02 m) consumed 2 28 mL d 1 of OliP or a taste- and appearance-matched placebo (PL) over 16 consecutive days. Participants completed a demanding, aerobic exercise protocol at ~75% maximal oxygen uptake ( . V O 2max) for 65 min 24 h before sub- and maximal performance exercise tests prior to and following the 16-day consumption period. OliP reduced the time constant ( ) (p= 0.005) at the onset of exercise, running economy (p =0.015) at lactate threshold 1 (LT1), as well as the rating of perceived exertion (p= 0.003) at lactate turnpoint (LT2). Additionally, OliP led to modest improvements in acute recovery based upon a shorter time to achieve 50% of the end of exercise . V O 2value (p= 0.02). Whilst OliP increased time to exhaustion (+4.1 1.8%), this was not signi cantly different to PL (p> 0.05). Phenolic compounds present in OliP, including HT and related metabolites, may provide bene ts for aerobic exercise and acute recovery in recreationally active individuals. Further research is needed to determine whether dose-response or adjunct use of OliP alongside longer-term training programs can further modulate exercise-associated adaptations in recreationally active individuals, or indeed support athletic performance. Keywords: polyphenols; OliPhenolia ® ; hydroxytyrosol; exercise; oxygen uptake kinetics; lactate threshold; running economy 1. Introduction Nutritional strategies to enhance exercise performance and recovery are of current scienti c interest to individuals who regularly undertake physical activity, competitive athletes, military workers, as well as the general population. Recent approaches which have gained popularity in an attempt to attenuate exercise-induced muscle damage (EIMD) and oxidative stress include the supplementation of naturally occurring phytochemicals (i.e., polyphenols) from sources such as pomegranate, cocoa, or cherries [1–3]. The average adult consumption of polyphenols is suggested to be ~1 g d 1 [4], with primary sources from fruits, vegetables, beverages such as tea and coffee, wine, and chocolate [5]. With antioxidant properties [6], nutritional polyphenols may act as radical scavengers and metal Nutrients2023,15, 421.

occurring phytochemicals (i.e., polyphenols) from sources such as pomegranate, cocoa, or cherries [1–3]. The average adult consumption of polyphenols is suggested to be ~1 g d 1 [4], with primary sources from fruits, vegetables, beverages such as tea and coffee, wine, and chocolate [5]. With antioxidant properties [6], nutritional polyphenols may act as radical scavengers and metal Nutrients2023,15, 421.

Nutrients2023,15, 421 2 of 20 chelators, regulating metabolism, body mass, chronic disease, and cell proliferation [7]. Free radicals and reactive oxygen and nitrogen species (RONS) are the primary oxidizing agents produced in cellular biochemical reactions for aerobic energy production [5]. Aerobic exercise is characterized by increased total energy expenditure [8], where the availability of endogenous substrates and aerobic metabolism are crucial for overall performance [9,10]. The increased oxygen (O2) demand by skeletal muscles during exercise results in greater free radical production and an increase in RONS [11]. Whilst viewed as detrimental to the cell for many years, recent evidence shows that RONS are crucial physiological activators and regulators of various intracellular signaling pathways in response to stress, enhanc- ing defense, improving cell adaptation, and upregulating the expression of endogenous antioxidant enzymes [12,13]. Furthermore, exercise adaptations are dependent, at least partially, on an acute ox- idative stress response. When exercise intensity is matched, individuals expressing lower levels of RONS have demonstrated inferior training adaptations compared to those with moderate or higher levels of RONS [14]. However, during excessive and demanding exer- cise, an imbalance between RONS and endogenous antioxidants induces oxidative damage, potentially impacting at a mitochondrial or DNA level [15], reducing vasodilatory capac- ity [16] and contractile force within the muscle through impaired calcium sensitivity [17]. This can have inferences for repetitive training sessions or longer-term adaptations and may, therefore, impair exercise performance and/or the recovery process.In sports where arterial blood flow and maximum cardiac output are determinants of performance (i.e., endurance and team-based sports), acute ingestion (<3 h before competition) or chronic supplementation of polyphenols (~7-days) could improve time to exhaustion at 70% maximum oxygen uptake ( . VO2max) by +9.7% [ The mechanisms by which polyphenols may facilitate ergogenic effects reportedly occur via nitric oxide synthase production [20] as well as the activation of sirtuin 1 (SIRT1) [21,22]. SIRT1 deacetylates several transcription factors such as forkhead (FOXO) proteins and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC- 1 ) [23]. This can facilitate mitochondrial biogenesis, endothelial function, cell proliferation and differentiation, metabolic ef ciency, resistance to stress, and improve

ergogenic effects reportedly occur via nitric oxide synthase production [20] as well as the activation of sirtuin 1 (SIRT1) [21,22]. SIRT1 deacetylates several transcription factors such as forkhead (FOXO) proteins and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC- 1 ) [23]. This can facilitate mitochondrial biogenesis, endothelial function, cell proliferation and differentiation, metabolic ef ciency, resistance to stress, and improve in ammatory and immune function [24–26]. The supplementation of phenolic compounds, and gut-derived metabolites, may therefore provide adjunct or indirect ergogenic effects on physical perfor- mance by way of potentially reducing the O2cost of exercise (i.e., economy), enhancing . V O2maxor exercise tolerance, and/or improving substrate utilization ef ciency. Previ- ous ndings have highlighted that polyphenol nutrients (e.g., resveratrol) may support mitochondrial function [27] and may therefore modulate essential biological functions (including thermogenesis, mitochondrial biogenesis and adenosine triphosphate produc- tion) [28]. These functions are pivotal for trained, recreationally active and untrained exercising individuals, ensuring that substrate supply kinetics and waste product removal match the requirements of the speci c exercise bout [29]. Furthermore, it could be inferred that due to the anti-in ammatory and immuno- modulatory effects of phenolic compounds, an increase in polyphenol consumption (from food or supplementation) may be pertinent to exercise recovery. A reduction in physio- logical stressors that negatively impact exercise training [30] may support fast and slow phases of recovery, in uencing performance in both prolonged or repeated bouts of exercise. Evidence for enhanced functional recovery from foods/supplements high in polyphenol compounds (e.g., Montmorency cherries > 5-days) have been exhibited in both trained and untrained individuals in a multitude of general exercise settings [31–33]. However, further research is warranted to investigate other polyphenols or novel food products, to assess markers of exercise recovery and identify the potential impact of phenolic compounds in speci c exercise settings. This is the rst study to undertake an investigation into a commercially available polyphenol-rich olive fruit water, OliPhenolia ® (OliP), which has not been assessed in an exercise domain. Originating during the olive picking season, this polyphenol-rich drink

the potential impact of phenolic compounds in speci c exercise settings. This is the rst study to undertake an investigation into a commercially available polyphenol-rich olive fruit water, OliPhenolia ® (OliP), which has not been assessed in an exercise domain. Originating during the olive picking season, this polyphenol-rich drink

Nutrients2023,15, 421 3 of 20 is extracted via concentration, reverse osmosis, and ceramic membrane technology at the aqueous part of the olive fruit. Whilst OliP contains a variety of phenolic compounds, it is particularly rich in hydroxytyrosol (HT).Abundant in olives in the form of pure HT, HT glycosides and oleuropein, HT is an effective antioxidant, with studies highlighting protection against oxidative stress in vascular tissue [34,35], low-density lipoprotein oxidation [36–38], and a reduction in oxidative damage in intestinal epithelial cells [39], hepatocytes, and erythrocytes [40]. However, OliP has yet to be considered within an exercise and/or recovery domain and thus, requires investigation. Therefore, this study investigated the effect of OliP on submaximal and exhaustive exercise, as well as respiratory markers of acute recovery, in recreationally active volunteers. Understanding the ef cacy of OliP may inform future nutritional strategies pertinent to exercise training and recovery. 2. Materials and Methods 2.1. Ethical Approval and Trial Registration This study was registered with clinical-trials.gov (ID: NCT04959006) with ethical approval obtained from the Faculty of Science and Engineering Research Ethics Panel, Anglia Ruskin University (Ethical approval no. FSE/FREP/20/946). Following a priori power calculation (G*power3, Dusseldorf, Germany [41]) using = 0.05 and 1- = 0.80, from previous reports of a time trial run and following recovery (plasma free radicals, post run pain and time to recovery [h]) [42], a minimum sample size of eight per intervention group was estimated. 2.2. Participant Characteristics Eligibility for the study required participants to be recreationally active (undertaking ~3 exercise sessions a week), with a . V O2maxof >25 mL kg 1 min 1 determined at the rst visit. All participants were >21 yrs, with no known metabolic disorders, viruses, or infec- tions; were not self-administering any polyphenol or antioxidant-rich supplementation or adhering to speci c diets that could con ict with study parameters. A total of 32 healthy participants volunteered and engaged with the study. However, following a review of indi- vidual protocol adherence and analysis of outliers, 3 participants' datasets were removed. General characteristics of the remaining 29 participants satisfactorily completing the study are displayed in Table. Table 1.

or adhering to speci c diets that could con ict with study parameters. A total of 32 healthy participants volunteered and engaged with the study. However, following a review of indi- vidual protocol adherence and analysis of outliers, 3 participants' datasets were removed. General characteristics of the remaining 29 participants satisfactorily completing the study are displayed in Table. Table 1. Mean standard error (SE) participant characteristics overall and for OliPhenolia ® (OliP) and placebo (PL) groups respectively. Variable Overall OliP PL (n= 29; 20 M, 9 F) (n= 15; 11 M, 4 F) (n= 14; 9 M, 5 F) Age (yrs) 42 2 42 3 42 3 Height (m) 1.76 0.02 1.77 0.03 1.75 0.03 Body mass (kg) 71.08 2.14 73.57 2.44 68.41 3.52 Fat free mass (kg) 57.67 2.31 59.33 3.05 55.89 3.56 Body mass index (kg m 2 ) 22.9 0.4 23.5 0.4 22.3 0.7 Body fat (%) 18.7 1.8 19.5 2.2 17.8 3.0 . VO 2max(L min 1 ) 3.53 0.16 3.56 0.22 3.49 0.24 . VO 2max(mL kg 1 min 1 ) 49.6 1.7 48.3 2.5 51.0 2.2 M = male; F = female; . V O2max= maximal oxygen uptake. No statistical differences were reported between groups (p> 0.05). 2.3. Experimental Design Using a randomized number generator process (www.randomizer.org; accessed on 10 May 2021), participants were allocated into two supplement intervention groups (OliP or PL) in a double-blind manner. All participants reported to the Cambridge Centre for Sport and Exercise Sciences (CCSES), Anglia Ruskin University, on ve separate occasions, the rst of which involved an initial familiarization session [43,44]. All laboratory visits were

Nutrients2023,15, 421 4 of 20 conducted at the same time of day following an overnight fast (~10 h), with participants arriving in a euhydrated state. Participants were instructed to avoid strenuous and/or excessive exercise for the 24 h prior to testing visits, as well as adhere to all dietary instruc- tions for the 3-days pre-testing (Section ).For the duration of the supplementation period, participants were asked to continue habitual exercise and diet regimes, ensuring each week was matched to the previous in terms of training load, caloric and macronutrient intake. 2.4. Dietary and Exercise Activity Monitoring Dietary and hydration intake was tracked via the use of a mobile based application (MyFitnessPal, Inc., San Francisco, CA, USA). Participants were provided with a personal login and guidance instructions to support detailed dietary tracking. Participants were required to record consumption of all food items and liquids for 3-days leading into each exercise test as part of this study, as well as across the 16-day intervention period [45] and were checked regularly by the same researcher for consistency throughout the intervention. A list of polyphenol-rich `foods to avoid' was also provided for participants to adhere to in the 3-days leading into each laboratory visit (see Supplementary Materials, Table S1). Participants were also required to complete a standardized, daily exercise activity diary for the 3-days prior to exercise trials and the duration of the 16 consecutive days supplementa- tion period, ensuring they were rested for the 24 h prior to each visit. Participants were requested to maintain habitual lifestyle and exercise patterns across the study, ensuring consistency across the 16-day period throughout the course of supplementation. Session type, mean session heart rate, exercise duration and perceived session exertion (using a standard 0–10 visual analogue scale) were recorded following the completion of each training session as reported elsewhere [45]. 2.5. Laboratory Procedures All tests took place under controlled environmental conditions (temperature: 19.6 0.3 C; barometric pressure: 1005.6 1.2 mBar; and relative humidity: 48.4 2.2%). Upon arrival, participants rested for 10 min in a seated position before assessment of blood pressure (Omron 750CP, Kyoto, Japan), body mass

were recorded following the completion of each training session as reported elsewhere [45]. 2.5. Laboratory Procedures All tests took place under controlled environmental conditions (temperature: 19.6 0.3 C; barometric pressure: 1005.6 1.2 mBar; and relative humidity: 48.4 2.2%). Upon arrival, participants rested for 10 min in a seated position before assessment of blood pressure (Omron 750CP, Kyoto, Japan), body mass (electronic scale, Seca, Hamburg, Germany), and height (Seca stadiometer, Hamburg, Germany). At rest (baseline) and throughout exercise, 20 L capilliarized ngertip blood samples were collected for the assessment of blood lactate and glucose (Biosen C Line EKF-diagnostic analyzer, Cardiff, UK). Heart rate (HR) data were recorded in 5 s intervals using a short-range telemetric monitor (Polar 810s, Polar T34 strap, Kempele, Finland). For the initial familiarization trial, body composition was also recorded using bioelectrical impedance for the indirect assessment of body fat percentage, fat-free mass, and fat mass (Tanita SC-330ST, Amsterdam, The Netherlands). Breath-by-breath pulmonary gas variables (volume of O2[ . V O2], volume of carbon dioxide [ . V CO2], minute ventilation [ . VE ], respiratory exchange ratio [RER], breathing frequency [BF] and tidal volume [TV]) were measured continuously via a metabolic cart (MetaLyzer 3B-R2, Cortex Ltd., Leipzig, Germany) using a suitable facemask for each participant (7600 face mask with headgear, Hans Rudolph, Shawnee, Kansas, USA). Prior to each test, the MetaLyzer was calibrated as per manufacturers' speci cations. All exercise testing was completed on a Quasar Med Treadmill (HP Cosmos, Nussdorf, Germany). 2.5.1. Experimental Protocols—Visit 1, 3 and 5 Exercise intensities were calculated using lactate pro les from the familiarization trial (visit 1) and remained consistent in visit 3 and 5. Visits 1, 3 and 5 consisted of a two-part graded exercise test [46,47] including: (1) a submaximal incremental protocol, with a 10 min recovery period; and (2) a maximal test to volitional exhaustion (Figure).

Nutrients2023,15, 421 5 of 20 3 min 10 min Ramp to volitional exhaustion Recovery B B B B 5 min 50 min 1 min 60% ∆ LT1−LT2 10% above LT2 LT1 speed 1 h 3 min 10 min Ramp to volitional exhaustion Recovery 24 h Visit 5: Repeat of visit 3 Visit 4: Repeat of visit 2 Visit 1: Familiarization session Visit 2: Demanding aerobic session Visit 3: Submaximal and performance test session 16-day intervention 24 h ≥3-days End of study Figure 1. Schematic of study protocol outlining the familiarization (visit 1), demanding aerobic session (visit 2) and submaximal and performance test session (visit 3). B = blood sample; LT1 = lactate threshold; LT2 = lactate turnpoint. 2.5.2. Submaximal and Performance Test Protocol The speed for the submaximal protocol was selected at a pre-de ned level and in- creased by 1 km h 1 every 4 min, with 3 min of running at a constant speed [46,47] followed by a 1 min break for capilliarized ngertip blood sample collection. The gradient was main- tained at 1% [48] with rating of perceived exertion (RPE; 0 10 scale) and HR assessed in the nal 30 s of each running stage. For the . V O2maxperformance test, speed was held con- sistent with gradient increasing by 1% per min, with RPE and blood lactate concentration (B[La]) obtained at the end of the test. Participants ran until volitional exhaustion (which determined time to exhaustion [TTE]), with standardized verbal encouragement provided towards the end of the test. 2.5.3. Determination of Physiological Parameters and Respiratory Kinetics Lactate threshold (LT1) was determined by an initial rise in B[La] above baseline [49], and lactate turnpoint (LT2) was determined by a sudden and sustained increase in B[La] [50].

Nutrients2023,15, 421 6 of 20 Mean and standard deviations (SD) of . V O2for the last 30 breaths of each increment were calculated. Values 4 SD were removed as outliers with all remaining breaths averaged [44]. Running economy was calculated in mL kg 1 km 1 [51], described in Equation (1) below: Economy= . VO2 mL kg 1 min 1 Speed(km h 1 )/60 (1) The . V O2kinetics for exercise (on-kinetics) and recovery periods (off-kinetics) were modelled and calculated using validated software VO2FITTING [52]. Errant breaths were omitted by only including those within . V O2local mean 4 SD. Subsequently, the individ- ual on-transient breath-by-breath . V O2responses were modelled using a mono-exponential model [52] described in Equation (2): . VO2(t) = . VO 2baseline+A 1 e t (2) where . V O2(t) represents the relative . V O2at the timet, A and are the amplitude and time constant ( ) of the fast . V O2component.The individual off-transient breath-by-breath . V O2 responses were modelled using the a mono-exponential model [52] described in Equation (3): . VO2(t) =EE . VO2 A 1 e t (3) where EE . V O2represents the relative end-exercise . V O2during the on-transient kinetics phase. During exercise (on-kinetics), O2de cit, . V O2demand and were estimated [53]. The acute recovery period in this study re ected the 10 min following the submaximal exercise protocol. Within this period, time to 50% (T50%) was determined by the recording of consistent breaths under 50% of the . V O2maxvalue reached [19]. . V O2maxwas determined from the highest . VO2values recorded over a 15-breath rolling average [54]. 2.5.4. Demanding Aerobic Session—Visit 2 and 4 Visit 2 and 4 involved 65 min of exercise, with an overall target exercise intensity of ~75% . V O2max, designed to elicit muscular oxidative stress [45]. Participants completed a 5 min warm-up at a speed corresponding to LT1. Exercise intensity then increased to speeds that corresponded with 60% of the difference between LT1 and LT2 (DLT1-LT2) for 50 min, before completing a maximum of 5 1 min intervals at a speed

The study explores the impact of a phytocomplex on aerobic exercise and recovery.