Numerous human observational studies report that high intakes of fruits and vegetables are linked with a lower risk of chronic human diseases including cancer, vascular disease and diabetes [1,2,3,4,5]. While, many global organisations (WHO) and national government initiatives encourage consumers to consume a minimum of five portions or 400 g of fruit and vegetables per day [6, 18], recommendations for intake vary globally. A recent meta-analyses of 95 prospective studies reported a significantly reduced relative risk for CVD, stroke, total cancer incidence and all-cause mortality with fruit and vegetables intakes in excess of 200 g daily . Critically, this meta-analysis, which analysed data from a much larger number of studies than previous investigations, reported a dose–response relationship for intake, disease and death, with consumption of 800 g of fruit and vegetables per day (10 portions) considered optimal .
Here, the average reported intake of fruits, juices and vegetables at the start of the intervention was approx. 240 g each day, substantially below the recommended daily intake. Data from national surveys worldwide indicate that a large proportion of the general populace does not meet the guideline intake for fruits and vegetables [6, 18]. In a postal survey of 1069 men and women (aged 20–88 years) in the USA, 45% reported eating no fruit and 22% ate no vegetables daily . More recent data from the NHANES survey (1988–2002) indicate that only 11% of American adults meet the current recommendations . Intake data gathered from several European national food surveys and compiled by the European Food Safety Authority (EFSA), show that fruit and vegetable consumption averages 386 g per person per day in Europe . However, of the 16 EU countries sampled, only 4 achieved the recommended daily intake of greater than 400 g of fruit and vegetables, with 75% of countries failing to meet the WHO and national guidelines. In good agreement with the findings from the current study, average consumption in the UK was 258 g per day . Whilst there has been a slight increase in reported fruit and vegetable consumption in the period since 2001, only a minority (approx. 30%) of the UK population are meeting dietary guidelines [9, 49]. Failure to eat sufficient fruits and vegetables in order to maintain good health and prevent disease has been estimated to contribute 2.4% directly to the estimated overall burden of disease across Europe .
Compliance in this study, measured by dietary reporting (check list and 4-day weighed intake dairies) and plasma nutrient levels, was good, with the two treatment groups well differentiated in terms of fruit and vegetable consumption. The average intake was increased by 5 portions to a maximum of eight servings each day in the treatment group 12 weeks into the intervention. Consuming additional fruit, vegetables and fruit juice had no effect on calculated total energy, total fat, carbohydrate, protein or fibre intake, and a limited and transient effect on specific carbohydrate and lipid intake [starch, non-starch polysaccharides (NSP), total cholesterol and polyunsaturated fatty acid (PUFA) intakes], indicating no substantial displacement of habitual foods from the diet of the participants. As expected, antioxidant nutrient (vitamin C, β-carotene), folate and potassium intake increased substantially following consumption of up to 8.4 portions of fruits, vegetables and fruit juices daily for 12 weeks, suggesting potential beneficial effects of the intervention. The changes in nutritional micro- and macro-nutrient intakes described here agree well with findings from similar intervention studies [19, 44, 51,52,53]. Vitamin C, total carotene, β-carotene, potassium and NSP intake were significantly increased in volunteers fed five portions of fruit and vegetables for 8 weeks [51, 53]. Feeding volunteers an additional six portions of fruits and vegetables for 6 weeks significantly increased vitamin C, total carotenes, folate and NSP intake, measured by 24-h dietary recall . Vitamin C intake was also increased in subjects asked to adhere to a Mediterranean style diet for 2 months .
Less positively, NMES and total sugar intake also increased significantly in this study, probably reflecting the increased intake of fruit and fruit juice in the treatment group. The potential deleterious impact of increased NMES, sugars and calories from consuming fruit juice (particularly 100% fruit juice) to excess, is of public interest, particularly with regard to childhood obesity [55, 56]. Similar changes in total sugar, NMES and dietary fructose, glucose, maltose and galactose intake in response to substantially increasing fruit, juice and vegetable consumption have been reported previously [51,52,53].
There was, in general, good agreement observed between calculated dietary intake and measured plasma nutritional biomarkers. Consuming approx. 8 portions of fruits, vegetables and fruit juices daily for 12 weeks significantly increased blood folate (plasma and whole blood), vitamin C, the carotenoids α- and β-carotene and lutein/zeaxanthin in healthy men and women who habitually consumed a diet low in these foods prior to intervention. Post-washout changes in circulating nutrient status are consistent with those described in other studies. Feeding subjects considered to be at risk of developing CVD up to 6 additional portions of fruits and vegetables for 18 weeks increased plasma folate, vitamin C, total flavonoid and carotenoid levels . As here, changes in circulating nutrients broadly reflected increased estimated intake . Similarly, plasma from participants fed the equivalent of 5 portions of fruits and vegetables per day in the form of soups, juices and “shots” prepared from carrot, tomato, red peppers, apple, strawberries, orange, banana and cherries for 4 weeks had elevated α-carotene and β-carotene levels. Total carotenoid and vitamin C intake  were also increased. In contrast to the findings from our study, plasma lycopene increased (31%). This probably reflects differences in foodstuffs consumed during the intervention and suggests that tomatoes may have been displaced from the participants’ diet in this study. In a similar study, plasma β-carotene was elevated in volunteers fed 600 g/day of fruits vegetables and orange juice for 4 weeks . However, plasma vitamin C was unchanged, possibly due to a shorter intervention and a more restricted variety of foods . Increasing blood and dietary folate intake is associated with a decreased risk of cancer, CVD and stroke [28, 58]. In this study, participants consuming up to 8 portions of fruits, juices and vegetables daily for 12 weeks reported significantly increased plasma and whole-blood folate (a marker of long-term folate intake). An improvement in folate status was similarly observed in healthy men and women consuming fruits and vegetables containing 350 μg of folate each day for 4 weeks . Conversely, subjects eating 200 g of fruits and vegetables with a folate content of approx. 40 μg/day for an equivalent time period did not significantly increase blood folate concentrations .
Increasing fruit and vegetable consumption in this study was not found to affect measures of plasma antioxidant capacity or cellular antioxidant function. Fruits and vegetables contain high levels of antioxidant vitamins and phytochemicals including phytophenols that enhance antioxidant status and oxidative stress resistance in vitro and in vivo [25,26,27]. Evidence, primarily from in silico and in vitro studies, has demonstrated these compounds to be powerful scavengers of ROS. Oxidative stress, which occurs when the formation of ROS and other radical species overwhelms the circulating and intracellular defence systems, is implicated in the development of several human chronic diseases including diabetes, CVD, stroke and cancer [1,2,3,4]. Here, antioxidant capacity (plasma HORAC, TEAC, and FRAP), antioxidant enzyme activity (red cell catalase, glutathione peroxidase and superoxide dismutase) were unaffected by intervention. Similarly, increasing fruit and vegetable consumption in excess of WHO and UK government recommendations, did not improve the resistance of lymphocytes isolated from the participants to an induced oxidative stress. The 4-day weighed intake data collected throughout this study, which showed a significant increase in reported fruit, juices and vegetable consumption of approximately 5 portions per day in the intervention group, together with the observed increases in circulating vitamins and carotenoids, indicate that the lack of effect of supplementation reported here was not a consequence of poor compliance (Fig. 1). Rather, it suggests that increasing fruit and vegetable consumption significantly increases the circulating concentrations of beneficial nutrients in healthy subjects without inducing a corresponding change in antioxidant capacity or markers of oxidative stress. The effect of consuming plant-based foods and beverages on plasma antioxidant capacity is inconsistent, despite significant conservation of the methodologies employed [57, 60].
A recent meta-analysis of more than 100 interventions found a generally positive effect of fruits, vegetables, plant foods, red wine and tea on plasma non-enzymatic antioxidant capacity (NEAC; ). Similarly, FRAP (which was used in this study) was elevated slightly (approx. 10%) in subjects at risk of CVD who consumed up to six additional portions of fruits and vegetables per day for up to 18 weeks . Eating a Mediterranean style diet for 8 weeks, while significantly increasing total estimated vitamin C intake (approx. 30%) in subjects with abdominal obesity, also caused a small increase in plasma ORAC (approx. 8%) and a trend towards increased FRAP activity . Equally, a null or highly limited effect of fruit and vegetable intervention on surrogate markers of disease risk has also been reported. Increasing fruit and vegetable intake had no effect on plasma antioxidant capacity (estimated by TEAC, FRAP, ORAC), erythrocyte SOD, catalase, glutathione reductase and GST activities (although GPX was significantly elevated), after consuming 600 g of fruit and vegetables per day for 24 days in a placebo-controlled intervention . Leucocyte DNA single-strand breakage, DNA base oxidation damage (pyrimidines and purines), sensitivity to DNA oxidation damage and DNA repair enzyme efficacy (hOGG1 and ERCCI) were also unchanged . A lack of effect on antioxidant capacity and cellular antioxidant activity was reported in a similar intervention (600 g of fruit and vegetables daily for 25 days), although biomarkers of protein and lipid oxidation did respond positively, indicating that specific molecular targets may react differently to intervention . Conversely, consuming a diet supplemented for 8 weeks with antioxidant-rich plant-based foods had a significant positive effect on DNA single-strand breakage (20% reduction) and base excision repair capacity (40% increase), in a group of healthy men. Here, oxidised DNA base damage and resistance to oxidative DNA damage was unchanged by the intervention, while, inexplicably, nucleotide excision repair activity was substantially decreased (39%; ).
Interestingly, consuming non-nutritional quantities of a single foodstuff, or fruits and vegetables rich in a particular nutrient has been shown to alter the status of several of the biomarkers employed here. Feeding healthy subjects a single meal (200 g) of cooked onions which are high in flavonoids, significantly reduced DNA oxidation damage in lymphocytes collected from the volunteers . Similarly, endogenous DNA damage and base excision DNA repair capacity were improved in lymphocytes from volunteers fed a large bolus of kiwi fruit puree (equivalent to three fruits) . It has been suggested that antioxidants delivered in fluid-based interventions using wine, fruit and vegetable juices and purees, are more bioavailable than in complex food matrixes. Drinking red wine (375 ml/day for 2 weeks) was found to increase total plasma phenolic concentration and reduce markers of oxidation (TBARS and conjugated dienes) in oxidised LDL from healthy subjects . Similarly, circulating vitamin C, α- and β-carotene were significantly higher post-supplementation in healthy participants fed an equivalent amount (400 g/day) of fruits and vegetables in liquid form compared with nutrient levels measured here . In agreement with the findings from this study, feeding a liquid diet was not associated with any improvement in biomarkers of DNA damage, inflammation or oxidative stress . Likewise, feeding healthy subjects 750 ml of cranberry juice daily for 2 weeks significantly increased plasma vitamin C concentrations (approx. 30%), but had no protective effect on blood antioxidant capacity (FRAP, catalase, SOD and GSHPx) or genomic stability (DNA oxidation damage in lymphocytes and urine) .
This study has limitations. Fasted blood was collected and it is possible that any bioefficacy of the fruit and vegetables consumed is short-lived and undetectable using the biomarkers employed here. Indeed, we have shown previously that ingesting cranberry juice significantly increases plasma antioxidant capacity over a few hours, but that this effect is transient . Moreover, certain of the assays employed here, while sensitive to changes in global antioxidant capacity are not relevant for measuring the impact of fruits and vegetables on systemic inflammation or vascular health (e.g. CRP, IL-6, sICAM, sVCAM and PAI-1). However, as described earlier, this study was not powered to identify changes in these biomarkers. A primary aim of this study was to investigate the impact of increasing fruit, juice and vegetable intake in an average normal population. Consequently, heathy subjects, with no pathologies and not taking prescribed medication were purposefully recruited. Antioxidant activity (NEAC) has been found to respond to intervention more strongly in subjects displaying chronic disease risk factors when compared with healthy subjects . Whether this intervention would have had a greater impact in subjects with a higher BMI or hypercholesterolemia/hyperhomocysteinemia and considered at greater risk of chronic diseases, remains to be established.
Consumption of fruits, vegetables and juices returned to almost baseline levels in the treatment group after the intervention. The slight increase reported was equivalent to only half of one portion in this group (2.63 portions increasing to 3.33 portions). This increase in intake agrees well with published studies, where the average improvement gained by interventions designed to foster a sustained increase in fruit and vegetable consumption was approximately one half serving more per day . However, this figure is substantially lower than that reported by Cox et al., who observed a sustained increase of 4.6 portions, 12 months after the end of an 8-week study . Long-term positive changes in fruit and vegetable consumption were also reported following a randomised controlled trial very similar in design to that described here . Here, participants were asked to consume less than 2 (control) or more than 5 (intervention) portions of fruits, vegetables and juices daily for 16 weeks. Participants in the intervention group increased their intake to an average of 6.0 portions of fruits, vegetables and juices each day at the end of the study . At follow-up, while fruit and vegetable intake in this group had dropped substantially to 3.6 portions/day, former volunteers were still consuming a significantly higher intake of fruits and vegetables 18 months after the intervention when compared with control participants (2.6 portions/day; ).
Despite evidence that the vast majority of consumers fail to meet national guidelines for fruit and vegetable intake, most people, including those in this study, are aware of government recommendations, believe that it is easy to eat a balanced heathy diet and, that as individuals, they do eat healthily [49, 66,67,68]. Here, all of the participants were aware of the “5 A Day” guidelines, and believed that it was important to increase their fruit and vegetable intake, indicating that nutritional knowledge is not in itself a barrier to consumption. The majority of participants in both groups reported liking fruits and vegetables post-intervention, although this was less evident in those that had received fruits and vegetables within the intervention. Critically, these participants perceived that it was more difficult to eat the recommended five servings per day than those in the control group who had maintained a habitual low intake of approx. 3 servings. This may be a reflection of having experienced eating in excess of eight portions a day compared with those in the control group. This agrees with earlier findings where participants reported greater difficulty in eating two servings of vegetables with a meal after they had taken part in a fruit and vegetable intervention [51, 67].
Primary reasons given for the reduction in intake in the treatment group post-study were that the amount of fruit and vegetables in the intervention had been too much to continue eating, that it was inconvenient to shop for fresh fruits and vegetables as frequently as required, and that it was too expensive to eat more than five serving of fruits and vegetables daily. It is worth remembering that all fruits and vegetables were provided cost-free and that food delivery was tailored as far as possible to the participants work schedule and availability throughout this study. Moreover, it is noteworthy that, once withdrawn (washout period) fruit and vegetable consumption quickly declined towards habitual intake levels. These findings should be incorporated in the design of future population-focused interventions to promote healthy eating.