This investigation examined the impact of a 1000 kcals·d-1 increase in exercise energy expenditure on NBAL and whole-body protein turnover in healthy male volunteers who differed in aerobic fitness level. The main objective of the study was to determine whether the effects of increased exercise on protein metabolism would be modulated by fitness level. We hypothesized that whole-body protein turnover would increase in both fit and unfit individuals in response to the increase in exercise energy expenditure, while daily NBAL would be negative initially and rise over time as nitrogen retention improved with adaptation to the increased exercise. We also thought that the increase in whole-body protein turnover would be attenuated, and NBAL would be preserved to a greater degree in FIT than LOW-FIT persons.
Contrary to our hypothesis, we found no significant changes in whole-body protein turnover in response to the increased exercise energy expenditure (i.e., no effect of time or group-by-time interaction). Further, when the whole-body protein turnover data for both groups was combined and analyzed using aerobic fitness level (VO2peak) as a covariate, no significant associations between VO2peak and markers of whole body protein turnover were observed. We did, however, observe a significant group effect as LOW-FIT had higher levels of net protein oxidation compared to FIT and FIT had higher levels of net balance compared to LOW-FIT. Additionally, we demonstrated similar changes (time-course and magnitude) in NBAL due to increased energy expenditure for FIT and LOW-FIT: NBAL was initially negative in response to the exercise intervention, but then increased as volunteers adapted to the additional exercise. Contrary to our hypothesis, however, this observation was not dependent on aerobic fitness level.
The NBAL response we observed is consistent with previous reports by Gontzea et al.  who demonstrated that when sedentary subjects initiated an exercise regime, and caloric intake was 10% greater than expenditure, NBAL went from positive to negative, but later returned to zero by day 15. In our investigation, FIT and LOW-FIT achieved neutral and positive NBAL, respectively, after only 7 days of exercise training, indicating an adaptation in protein metabolism to the increased exercise and that protein intake was sufficient when subjects maintained energy balance. Indeed, although percent of energy contributed from protein was slightly below the lower level of the Acceptable Macronutrient Distribution Range , for both groups, protein intake calculated per kg body weight per day was adequate according to current recommendations .
Contrary to our expectations, we observed a negative nitrogen balance in the FIT group during the baseline period. It is possible that the 3- to 5-d adaptation period used in this investigation may not have been of sufficient duration to achieve a true adaptation to the protein level provided during the study for the FIT volunteers who habitually consumed ~1.6 g of protein·kg-1·d-1, which we previously acknowledged . Although the major initial changes in nitrogen excretion occur within approximately 5–7 days in adults according to a World Health Organization (1985) report , others have suggested a longer adaptation period may be necessary depending on the discrepancy between the habitual and "new" protein intake . Additionally, the level of dietary control during the adaptation period could also have impacted our baseline nitrogen balance values. Volunteers were still free-living during this adaptation period, and food was not provided, therefore, it is possible that they may not have adhered to the run-in diet closely enough.
Despite the initial negative nitrogen balance observed in the FIT group, volunteers still achieved NBAL after 7 days of exercise training and our NBAL results are consistent with reports by Butterfield and Calloway  and Todd et al. , showing that adequate energy intake maintained NBAL in the face of an unaccustomed increase in exercise energy expenditure. Indeed, we previously reported that fit volunteers consuming 1.0 g protein·kg-1·d-1, who did not match their energy intake with this increased energy expenditure were in a state of negative NBAL throughout this 7-d period .
Our results demonstrate a significant between group effect for whole-body protein turnover, regardless of time. The fact that we observed a higher net protein oxidation in LOW-FIT compared to FIT independent of time might have been expected, given the decrease in protein oxidation that was observed in response to endurance training both at rest  and during exercise . Our results demonstrating a less negative net balance for FIT compared to LOW-FIT independent of time is also not surprising, given that trained persons are more efficient at protein metabolism [3–5]. We also observed a tendency (P = 0.06) for higher whole-body protein synthesis in FIT compared to LOW-FIT independent of time, and that has not been reported before. Observed differences in net protein balance between the NBAL and tracer methodology (that is, net balance remained negative whereas NBAL shifted to neutral or positive) are likely due to the fact that tracer measures were performed in the fasted state while NBAL included fasted and fed periods. Although measurements of whole body protein metabolism may not be reflective of changes in fractional synthetic rate at the muscle level, this may provide insight into our findings since fractional synthetic rate in the muscle has been reported to increase in response to chronic aerobic [7, 8] and resistance exercise training .
Whole-body protein turnover responses to an acute (1–7 days) increase in exercise energy expenditure of the magnitude employed in this study (1000 kcals·d-1) has not been investigated in either sedentary or aerobically fit individuals. Contrary to our hypothesis, our whole-body protein turnover results do not demonstrate a significant time effect or group-by-time interaction, in response to an increase in aerobic energy expenditure. One possible explanation for why differences were not observed may be related to different protein intakes between groups. While protein intake was matched for grams·kg body weight-1·day-1, differences in FFM between the groups resulted in a statistically significant difference in grams of protein·kg FFM-1·day-1 (i.e. protein intake was higher for LOW-FIT vs. FIT). However, we contend that it is unlikely that this difference between groups (< 1.0 g protein per day) would elicit a true physiological response and confound our findings. However, the reality that FIT had to adapt to the "new" protein intake, in addition to the increase in exercise energy expenditure, may have masked any group-by-time differences in whole-body protein turnover responses.
It is probably inappropriate to compare our results to studies employing chronic aerobic training, since those studies sought to elucidate "training adaptations" in regards to whole-body protein turnover. We did not expect to elicit adaptations over the course of 7 days, but sought to determine if aerobic fitness level exerted a protective effect on protein utilization and protein balance when individuals were in a catabolic state. In any event, the changes reportedly occurring in response to experimental aerobic training programs are inconsistent, with some reporting no change in resting leucine oxidation [6, 8] and another suggesting a decrease in leucine oxidation .
In terms of the tracer measurements, McKenzie et al  observed an improvement in protein utilization when leucine oxidation was measured during exercise, but no change in protein utilization when leucine oxidation was measured at rest in response to 38 days of endurance training. Therefore, we may have detected group-by-time interactions if we had measured protein utilization during, or immediately after exercise, instead of at rest. Additionally, the use of multiple tracers to more accurately assess whole-body protein turnover, particularly the addition of a branched-chain amino acid tracer such as leucine, may have yielded differential results since the metabolism of one particular amino acid may not be representative of all the amino acids in the body . We chose phenylalanine as a tracer because it is not synthesized endogenously or oxidized by muscle. Additionally, results derived from this method have been shown to be similar to those from the leucine method . This approach is not without its weaknesses [25, 26], however, which we previously acknowledged .
The fitness level of our untrained volunteers is perhaps another reason why we did not detect differences in whole-body protein turnover between trained and untrained volunteers in response to the increased exercise. Our population of low-fit volunteers routinely expended ~3000 kcals·d-1 and increased to ~4000 kcals·d-1 during the intervention. Therefore, they perhaps should be characterized as "moderately active". This is in contrast to the 'sedentary' volunteers utilized in other acute exercise trials similar to ours who regularly expended an average of ~2000 kcals·d-1 during baseline testing and ~3000 kcals·d-1 during the exercise intervention . Therefore, our volunteers do not fit the traditional definition of "sedentary", and perhaps the moderate activity demonstrated by our "low-fit" volunteers is enough to elicit "training adaptations" that spare body nitrogen and attenuate protein utilization. In order to further elucidate this notion, we combined the data from both groups and conducted repeated measures ANOVA with aerobic fitness level (VO2peak) as a covariate. Indeed, for whole-body protein synthesis and derived whole-body protein breakdown, the range in VO2peak was not large enough between volunteers to significantly influence these outcome measures. However, when the range in VO2peak was large enough to influence net protein oxidation and derived net balance, no significant associations were observed.
Despite the potential limitations presented herein, this rigorously controlled study provides insight into the notion that aerobic fitness level modulates the effects of increased exercise on protein metabolism. Similar to other investigations, our findings demonstrate that aerobic fitness level modulates whole-body protein turnover at rest regardless of the unaccustomed increase in exercise. However, our results did not reveal any significant changes in resting measures of whole-body protein turnover between fit and low-fit males in response to the increased exercise, nor was there any substantial evidence that aerobic fitness level influences net protein oxidation or derived net balance in response to increased exercise. These findings suggest that chronic aerobic training does not impart metabolic adaptation to spare body protein in response to an unaccustomed increase in energy expenditure when measured at rest.