This is the first meta-analysis to directly investigate the effects of protein timing on strength and hypertrophic adaptations following long-term resistance training protocols. The study produced several novel findings. A simple pooled analysis of protein timing without controlling for covariates showed a significant effect on muscle hypertrophy (ES = 0.24 ± 0.10) with no significant effect found on muscle strength. It is generally accepted that an effect size of 0.2 is small, 0.5 is moderate, and 0.8 and above is a large, indicating that the effect of protein timing on gains in lean body mass were small to moderate. However, an expanded regression analysis found that any positive effects associated with protein timing on muscle protein accretion disappeared after controlling for covariates. Moreover, sub-analysis showed that discrepancies in total protein intake explained the majority of hypertrophic differences noted in timing studies. When taken together, these results would seem to refute the commonly held belief that the timing of protein intake in the immediate pre- and post-workout period is critical to muscular adaptations [3–5].

Perceived hypertrophic benefits seen in timing studies appear to be the result of an increased consumption of protein as opposed to temporal factors. In our reduced model, the amount of protein consumed was highly and significantly associated with hypertrophic gains. In fact, the reduced model revealed that total protein intake was by far the most important predictor of hypertrophy ES, with a ~0.2 increase in ES noted for every 0.5 g/kg increase in protein ingestion. While there is undoubtedly an upper threshold to this correlation, these findings underscore the importance of consuming higher amounts of protein when the goal is to maximize exercise-induced increases in muscle mass. Conversely, total protein intake did not have an impact on strength outcomes and ultimately was factored out during the model reduction process.

The Recommended Dietary Allowance (RDA) for protein is 0.8 g/kg/day. However, these values are based on the needs of sedentary individuals and are intended to represent a level of intake necessary to replace losses and hence avert deficiency; they do not reflect the requirements of hard training individuals seeking to increase lean mass. Studies do in fact show that those participating in intensive resistance training programs need significantly more protein to remain in a non-negative nitrogen balance. Position stands from multiple scientific bodies estimate these requirements to be approximately double that of the RDA [59, 60]. Higher levels of protein consumption appear to be particularly important during the early stages of intense resistance training. Lemon et al. [61] displayed that novice bodybuilders required a protein intake of 1.6-1.7 g/kg/day to remain in a non-negative nitrogen balance. The increased protein requirements in novice subjects have been attributed to changes in muscle protein synthetic rate and the need to sustain greater lean mass rather than increased fuel utilization [62]. There is some evidence that protein requirements actually decrease slightly to approximately 1.4 g/kg/day in well-trained individuals because of a greater efficiency in dietary nitrogen utilization [63], although this hypothesis needs further study.

The average protein intake for controls in the unmatched studies was 1.33 g/kg/day while average intake for treatment was 1.66 g/kg/day. Since a preponderance of these studies involved untrained subjects, it seems probable that a majority of any gains in muscle mass would have been due to higher protein consumption by the treatment group. These findings are consistent with those of Cermak et al. [24], who found that protein supplementation alone produced beneficial adaptations when combined with resistance training. The study by Cermak et al. [24] did not evaluate any effects regarding timing of intake, however, so our results directly lend support to the theory that meeting target protein requirements is paramount with respect to exercise-induced muscle protein accretion; immediate intake of dietary protein pre and/or post-workout would at best appear to be a minor consideration. The findings also support previous recommendations that a protein consumption of at least 1.6 g/kg/day is necessary to maximize muscle protein accretion in individuals involved in resistance training programs [61].

For the matched studies, protein intake averaged 1.91 g/kg/day versus 1.81 g/kg/day for treatment and controls, respectively. This level of intake for both groups meets or exceeds suggested guidelines, allowing for a fair evaluation of temporal effects. Only 3 studies that employed matched protein intake met inclusion criteria for this analysis, however. Interestingly, 2 of the 3 showed no benefits from timing. Moreover, another matched study actually found significantly greater increases in strength and lean body mass from a time-divided protein dose (i.e. morning and evening) compared with the same dose provided around the resistance training session [19]. However, this study had to be excluded from our analysis because it lacked adequate data to calculate an ES. The sum results of the matched-protein studies suggest that timing is superfluous provided adequate protein is ingested, although the small number of studies limits the ability to draw firm conclusions on the matter.

This meta-analysis had a number of strengths. For one, the quality of studies evaluated was high, with an average PEDro score of 8.7. Also, the sample was relatively large (23 trials encompassing 478 subjects for strength outcomes and 525 subjects for hypertrophy outcomes), affording good statistical power. In addition, strict inclusion/exclusion criteria were employed to reduce the potential for bias. Combined, these factors provide good confidence in the ability draw relevant inferences from findings. Another strength was the rigid adherence to proper coding practices. Coding was carried out by two of the investigators (BJS and AAA) and then cross-checked between coders. Coder drift was then assessed by random selection of studies to further ensure consistency of data. Finally and importantly, the study benefited from the use of meta-regression. This afforded the ability to examine the impact of moderator variables on effect size and explain heterogenecity between studies [64]. Although initial findings indicated an advantage conferred by protein timing, meta-regression revealed that results were confounded by discrepancies in consumption. This ultimately led to the determination that total protein intake rather than temporal factors explained any perceived benefits.

There are several limitations to this analysis that should be taken into consideration when drawing evidence-based conclusions. First, timing of the meals in the control groups varied significantly from study to study. Some provided protein as soon as 2 hours post workout while others delayed consumption for many hours. A recent review by Aragon and Schoenfeld [23] postulated that the anabolic window of opportunity may be as long as 4–6 hours around a training session, depending on the size and composition of the meal. Because the timing of intake in controls were all treated similarly in this meta-analysis, it is difficult to determine whether a clear anabolic window exists for protein consumption beyond which muscular adaptations suffer.

Second, the majority of studies evaluated subjects who were inexperienced with resistance exercise. It is well-established that highly trained individuals respond differently to the demands of resistance training compared with those who lack training experience [65]. In part, this is attributed to a “ceiling effect” whereby gains in muscle mass become progressively more difficult as a trainee gets closer to his genetic hypertrophic potential. There also is emerging evidence showing that regimented resistance exercise attenuates anabolic intracellular signaling in rodents [66] and humans [67], conceivably diminishing the hypertrophic response. Our sub-analysis failed to show an interaction effect between resistance training status and protein timing for either strength or hypertrophy. However, statistical power was low because only 4 studies using trained subjects met inclusion criteria. Future research should therefore focus on determining the effects of protein timing on muscular adaptations in those with at least 1 year or more of regular, consistent resistance training experience.

Third, in an effort to keep our sample size sufficiently large, we pooled CSA and FFM data to determine hypertrophy ES. FFM is frequently used as a proxy for hypertrophy, as it is generally assumed that the vast majority of the gains in fat free mass from resistance training are myocellular in nature. Nevertheless, resistance exercise also is associated with the accretion of non-muscle tissue as well (i.e. bone, connective tissue, etc.). To account for any potential discrepancies in this regard, we performed sub-analyses on CSA and FFM alone and the results essentially did not change. For FFM, the difference between treatment and control was not significant (P = 0.27), with a ES difference of -0.08. Protein intake again was highly significant, with an ES impact of ~0.2 per every 1 g/kg/day. For CSA, the difference between treatment and control was not significant (P = 0.37), with a ES difference of -0.14. Protein intake was again significant (P = 0.02) with an ES impact of ~0.33 per every 0.5 g/kg.

Finally and importantly, there was a paucity of timing studies that attempted to match protein intake. As previously discussed, our results show that total protein intake is strongly and positively associated with post-exercise gains in muscle hypertrophy. Future studies should seek to control for this variable so that the true effects of timing, if any, can be accurately assessed.

Practical applications

In conclusion, current evidence does not appear to support the claim that immediate (≤ 1 hour) consumption of protein pre- and/or post-workout significantly enhances strength- or hypertrophic-related adaptations to resistance exercise. The results of this meta-analysis indicate that if a peri-workout anabolic window of opportunity does in fact exist, the window for protein consumption would appear to be greater than one-hour before and after a resistance training session. Any positive effects noted in timing studies were found to be due to an increased protein intake rather than the temporal aspects of consumption, but a lack of matched studies makes it difficult to draw firm conclusions in this regard. The fact that protein consumption in non-supplemented subjects was below generally recommended intake for those involved in resistance training lends credence to this finding. Since causality cannot be directly drawn from our analysis, however, we must acknowledge the possibility that protein timing was in fact responsible for producing a positive effect and that the associated increase in protein intake is merely coincidental. Future research should seek to control for protein intake so that the true value regarding nutrient timing can be properly evaluated. Particular focus should be placed on carrying out these studies with well-trained subjects to better determine whether resistance training experience plays a role in the response.