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Original Research

Blood Ammonium and Lactate Accumulation Response to Different Training Protocols Using the Parallel Squat Exercise

Rogatzki, Matthew J.1; Wright, Glenn A.1; Mikat, Richard P.1; Brice, A. Glenn2

Author Information
Journal of Strength and Conditioning Research: April 2014 - Volume 28 - Issue 4 - p 1113-1118
doi: 10.1519/JSC.0b013e3182a1f84e
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The purpose of exercise training is to challenge the body's physiological systems and provide a stimulus for adaptations to occur. Endurance and interval exercise is commonly monitored by blood analysis; however, blood analysis is used less often with resistance exercise. Currently, an accepted method for monitoring resistance exercise does not exist (15). Ammonium (NH4) and lactate (La) accumulation in the blood have been considered viable candidates for analysis (1,23) and deserve greater investigation.

The cause of blood NH4 and La accumulation during exercise has been established but not adequately examined in response to resistance exercise. Production of NH4 in the purine nucleotide cycle (PNC) indicates a rapid rate of adenosine triphosphate (ATP) hydrolysis (26), utilization of phosphocreatine (PCr) (26), and depletion of the total adenine nucleotide (TAN) pool (7). Lactate is produced at the end of glycolysis and represents muscle glycogen depletion and a decrease in pH (5). Because NH4 and La represent different energy systems, both metabolites may be useful in monitoring skeletal muscle metabolism.

Abernethy and Wehr (1) studied NH4 and La as an indicator of leg press exercise intensity. Blood NH4 and La levels were measured after the first and third set of leg presses performed at 15 repetition maximum (RM) and 5RM loads with 6 minutes of rest between sets for both protocols (1). They found that accumulation of NH4 was greatest during the 15RM protocol, confirming their hypothesis that the 3 × 15 RM would stimulate the PNC greater than 3 × 5 RM (1). Lactate increased with each measurement for both the 15RM and 5RM intensities; however, a parallel relationship between NH4 and La did not exist for either condition (1).

Abernethy and Wehr (1) intended on “understanding the metabolism associated with different repetition structures used in bouts of resistance activity … (to) allow strength and conditioning specialists, coaches, and sport scientists to better tailor resistance activity prescriptions to meet each individual's needs.” However, assessing the metabolic response to resistance training without the manipulation of training variables (sets, repetitions, rest time between sets) does not shed light on the effects of prescribed training sessions. To compare metabolic responses with an eye toward practical prescriptions for resistance training, NH4 and La should be measured after different types of workouts used in the field (muscle endurance, ME; hypertrophy, HYP; strength, STR) with equal work volumes. Without controlling for work volumes, a great amount of NH4 and La data variability may occur (1).

The purpose of our study was to analyze the accumulation of blood NH4 and La among resistance training protocols commonly used to develop ME, HYP, and STR with equal work volumes across trials. This will help scientists and strength and conditioning professionals assess the prescription of, and recovery from, such training programs. Because it is apparent that NH4 “increases in response to heavy resistance exercise” (23) and there appears to be a relationship between NH4 and La (12), our hypothesis is the STR protocol will cause the greatest blood NH4 and La levels followed by HYP and ME.


Experimental Approach to the Problem

To assess metabolic differences among ME, HYP, and STR protocols plasma NH4 and blood La concentration were measured before and after the squat exercise. A counterbalanced repeated measures design was used as each subject was their own control. We used the parallel back squat as a representative exercise because it is a lift common to resistance training programs, incorporates a large muscle mass, and will recruit motor units and fiber types across the continuum at the various loads incorporated in this study. Resistance training protocols designed to develop ME, HYP, and STR with equal work volumes were used, as these protocols are commonly used by strength and conditioning professionals (13,19,27).


Sixteen men from college (aged 18–24 years) volunteered to participate in this study. Men were used as subjects because of menstrual cycle effects on NH4 levels in women (7). We recruited subjects currently using the squat in their resistance exercise program and able to squat at least 1.5 times their body weight to ensure recruitment of those experienced with the squat. This study was undertaken halfway through spring semester allowing the subjects time to solidify a consistent workout routine. None of the participants reported using performance enhancing supplementation or drugs. Subjects were asked to follow a consistent diet based on their individual eating habits for the 12 hours before each session. Every subject recorded what he ate before the 1RM test and was reminded to eat a meal similar in content and volume before each test. This was required to control for nutritional intake and hydration levels. Testing sessions occurred at relatively the same time of day to control for prior sleep and natural diurnal rhythms. The protocols and methods of this study were approved by the University's Institutional Review Board for the Protection of Human Subjects. Before participation, each subject gave written informed consent.


Subjects visited the laboratory for 4 testing sessions. The initial session assessed each subject's 1RM for the squat, which was used to determine load in the subsequent testing sessions. Squat depth was visually analyzed by the same investigator to ensure that each subject achieved parallel (top of quadriceps parallel to the floor) for all testing sessions. Training variables were based on typical workout recommendations for ME, HYP, and STR (13,19,27). Greater than 3 but less than 7 days separated exercise testing sessions and protocol order was randomized among subjects to control for muscle adaptation and learning effects.

Workouts for ME consisted of 2 sets of 20 repetitions (2 × 20) at 53% of 1RM with a 45-second rest period between sets (13,19,27). The HYP workout consisted of 3 × 10 at 70% 1RM with a 120-second rest period between sets (13,19,27). For STR, workouts were 5 × 5 at 85% 1RM with a 180-second rest period between sets (13,19,27). All workouts for each subject were normalized to the same volume (p = 0.98) using the equation: sets × repetitions × external load = work volume.

Biochemical Assays

Blood was drawn (3 ml) before and after exercise from the antecubital vein into a heparinized vacuum tube. Subjects sat for 5 minutes before each draw because blood La and plasma NH4 concentrations have been found to peak around 5 minutes post exercise (4). All samples were immediately placed into an ice bath until centrifugation, which was performed within 30 minutes of the blood draws (11). Immediately before centrifugation, 1 ml of whole blood was removed from the heparinized test tube, placed into 2 ml of 70% perchloric acid (PCA) (Sigma No. 311421; Sigma-Aldrich, St. Louis, MO, USA) and frozen for future enzymatic analysis of La concentration. The remaining sample then underwent centrifugation (5,000 rpm for 5 minutes) to separate the plasma, which was immediately used for enzymatic determination of NH4 concentration. Standard precautions for handling blood NH4 specimens were taken as indicated by Huizenga et al. (11).

Plasma NH4 concentration was determined by adding 10 μl of plasma to 1 ml of ammonia reagent (Sigma No. A0853; Sigma-Aldrich), and then adding 10 μl of glutamate dehydrogenase (GLDH) (Sigma No. G2626; Sigma-Aldrich). After adding the GLDH, the decrease in absorbance was measured at 340 nm using a Spectronic 20D+ spectrophotometer (Thermo Electron Scientific Instruments LLC, Madison, WI, USA). Ammonium values were then determined by the following equation:

, where

= change in absorbance, TV = total assay volume, MW = molecular weight of NH4 (17 g/mole), F = dilution factor, [Latin Small Letter Open E] = extinction coefficient for NADPH at 340 nm, d = light path, and SV = sample volume (Ammonia Assay Kit AA0100, Technical Bulletin; Sigma-Aldrich). The Intra-coefficient of variation for NH4 analysis was 0.76%.

Blood La concentration was measured from whole blood samples by first adding 1 ml of whole blood to 2 ml of 70% PCA (Sigma No. 311421; Sigma-Aldrich), inducing red blood cell lysis. After red blood cell lysis, the mixture underwent centrifugation (5,000 rpm for 5 minutes), and 25 μl of the supernatant was added to a 0.8 M hydrazine and 1.0 M glycine buffer, pH 9.2 (Sigma No. 216046 and G7126; Sigma-Aldrich, respectively). After the supernatant and hydrazine-glycine buffer were mixed, 2.5 mg of nicotinarnide adenine dinucleotide (Sigma No. N7004; Sigma-Aldrich), and 50 μl of lactate dehydrogenase (LDH) (Sigma No. L2625; Sigma-Aldrich) were added. The increase in absorbance was then measured at 340 nm using a Spectronic 20D+ spectrophotometer (Thermo Electron Scientific Instruments LLC). Lactate values were found by using the formula

, where

= change in absorbance, PCAB = volume of blood plus PCA mixture, TV = total volume, [Latin Small Letter Open E] = extinction coefficient for NADH at 340 nm, d = light path, VB = volume of blood added to PCA, and SV = sample volume (Enzymatic Assay of l-Lactic Dehydrogenase; Sigma-Aldrich). The intra-coefficient of variation for La analysis was 4.92%. It is likely that the intra-coefficient of variation for La was much higher than for NH4 because absorbance readings for La being closer to 0.000 (average of 0.066) compared with NH4 (average of 0.431). The larger intra-coefficient of variation is likely not because of differences in pipetting technique because the same person was responsible for running both the NH4 and La assays.

Statistical Analyses

Differences among the dependent variables (work volume, La, and NH4) with means from the 3 protocols were assessed by 1-way repeated measures analysis of variance with a Greenhouse-Geisser correction when the assumption of sphericity was not met (25). Bonferroni pairwise comparisons were used when significant differences were present among protocols (17). Relationship between the rise of blood NH4 and La was determined by means of Bivariate Pearson's Correlation Coefficient (20). A 95% confidence interval (CI) was used to assign bounds of expected discrepancy between the sample mean and the population mean (6). All data are reported as mean (SD). Alpha was set at 0.05, and all statistics were generated using SPSS (Version 17; SPSS Inc., Chicago, IL USA).


Volume of work did not vary significantly among protocols (p = 0.98;

; observed power = 0.053) (STR, 6960.9 kg [SD = 1093.3], 95% CI: 6401.1–7520.8), (HYP 6881.3 kg [SD = 1115.6], 95% CI: 6321.4–7441.1), (ME 6912.5 kg [SD = 1126.6], 95% CI: 6352.6–7472.4). Average change of plasma NH4 levels from rest to postexercise was different among protocols (p = 0.001;

; observed power = 0.940) (Figure 1). The ME protocol caused a greater change in plasma NH4 (79.8 μM [SD = 45.4], 95% CI: 55.7–104.0) than both HYP (45.3 μM [SD = 34.5], 95% CI: 26.9–63.6, p = 0.017) and STR (31.7 µM [SD = 52.3], 95% CI: 3.9–60.0, p = 0.006) protocols. However, there was no significant difference found between the HYP and STR protocols (p = 0.85).

Figure 1
Figure 1:
Change of plasma NH4 from pre- to postexercise. Asterisks represent statistical difference (*p ≤ 0.05) compared with ME. Data presented as mean (SD). STR = strength; HYP = hypertrophy; ME = muscular endurance.

Average change of La among protocols was also found to be significantly different (p = 0.015;

; observed power = 0.741) (Figure 2). A significant difference (p = 0.005) existed for the average change of La concentration from rest to post exercise between the ME (6.1 mM [SD = 2.9], 95% CI: 4.6–7.7) and STR (3.9 mM [SD = 2.5], 95% CI: 2.6–5.2) protocols. No difference was found between the HYP (4.9 mM [SD = 2.5], 95% CI: 3.6–6.3) protocol and the STR (p = 0.491) or ME (p = 0.176) protocols. A moderate (r = 0.59), yet significant (p < 0.01) relationship between the NH4 and La response was found among protocols.

Figure 2
Figure 2:
Change of blood La from pre- to postexercise. Asterisks represent statistical difference (*p ≤ 0.05) compared with ME. Data presented as mean (SD). STR = strength; HYP = hypertrophy; ME = muscular endurance.


Our study found resistance exercise protocols consisting of high repetition and low load cause greater NH4 and La levels than protocols with lower repetitions and higher loads. This finding refutes our hypothesis of the STR protocol causing the greatest amount of NH4 and La production. We also discovered a nonparallel rise in NH4 and La levels indicating that differences of energy system involvement among the protocols does exist. Results from this investigation are novel, as to our knowledge, no other study has compared NH4 and La response to commonly used resistance training protocols.

The purpose of this study was to determine the metabolic response of resistance exercise by combining program variables the way that they are typically prescribed in the field (13,19,27). Time to complete the same amount of work differed among protocols. Total time for completion of each protocol, including rest between sets, was estimated to be approximately 233 seconds for ME, approximately 351 seconds for HYP, and approximately 828 seconds for STR. The time variables and dominant muscle fiber type associated with each protocol is likely the cause for differences in metabolic response.

Varying rest time between sets of the protocols likely played a role in NH4 and La differences. Rest between work sets of high-intensity exercise allow levels of PCr to be replenished by means of the phosphagen system using ATP generated from aerobic metabolism (3). Rest between sets for both STR (180 seconds) and HYP (120 seconds) likely allowed an adequate amount of time between sets for PCr resynthesis (8). However, the short rest time (45 seconds) between sets for the ME protocol likely prevented adequate PCr resynthesis before the commencement of the next set (8). Loss of immediate ATP resynthesis by means of PCr causes increased adenosine diphosphate (ADP) concentration leading to activation of the adenylate kinase reaction and a reduced TAN pool, indicated by rising NH4 levels (12). Accumulation of ADP, inorganic phosphate (Pi), and adenosine monophosphate (AMP) (5) during rapid ATP hydrolysis stimulates glycolysis causing depleted muscle glycogen, higher lactate levels, and decreased pH (5). If recovery between sets is short, as in the ME protocol, the resulting low levels of PCr will likely contribute to an increase of NH4 and La.

The heterogeneity in load and time under tension play a significant role in the production of NH4 and La during resistance exercise. As loads increase, there is a greater proportion of Type II motor units recruited (10). Greater activity of the PNC (16) and glycolytic (22) enzymes are found in Type IIx than IIa and less in Type I muscle fibers. Fewer repetitions at high load, as in the HYP and STR protocols, likely activate more Type II fibers than the ME protocol (10), but the low repetitions may not allow sufficient time under tension, approximately 111 and 108 seconds, respectively, for extended activation of the PNC and glycolysis (5,12). The longer time under tension for ME (approximately 188 seconds) likely allowed ample accumulation of ADP, AMP, and Pi for longer activation of PNC and glycolytic pathways leading to higher levels of NH4 (12,14) and La (9,21).

Parallel accumulation of NH4 and La has been attributed to the PNC and glycolysis both being stimulated by a high turnover rate of ATP and low levels of PCr (12). Existence of a parallel relationship would suggest that only NH4 or La would need to be measured, as one would be predictable of the other. Our study does not indicate a strong parallel accumulation between NH4 and La among resistance exercise protocols, shown by the moderate, albeit significant, correlation (r = 0.59) between the 2 variables. Our results are supported by Triplett et al. (24) who also found “low to moderate correlations” between La and NH4 at different time points after 5 sets of 10 repetitions of the parallel squat using 90% 1RM. Low to moderate correlation implies that although the production of NH4 and La may be kindled by similar factors, such as rapid ATP degradation, their respective accumulation in the blood may not be caused by the same intramuscular mechanisms. Abernethy and Wehr (1) also did not find a parallel increase between NH4 and La for the 15RM or 5RM protocols. These authors (1) demonstrated that higher repetitions per set lead to a greater accumulation of NH4 and La during resistance exercise on a leg press machine. However, this could indicate that accumulation of these metabolites is because of greater work volume. Using equated work volume across protocols in our study adds to the results of Abernethy and Wehr (1) by indicating that number of repetitions per set is important in determining metabolic stress.

Behm et al. (2) compared the effect of a single set of 5, 10, and 20 RM loads on isometric force production, muscle activation, and temporal twitch properties (peak force, time-to-peak force, and ½ relaxation time). They found that twitch contractile properties were affected to a greater degree by the higher RM protocols with no differences in muscle activation among protocols. The authors suggest at least a partial explanation may include that twitch properties are sensitive to decreases in pH along with increases in ADP and La in the muscle (2). These conditions are exacerbated as the number of repetitions per set increase (2). Other metabolic events that could lead to muscle fatigue include a decrease in PCr and TAN, leaving a lower energy state in the muscle. A rise in NH4 levels is caused by stimulation of the PNC by means of increased ADP accumulation, likely as a result of low levels of PCr. Lactate rises in response to stimulation of phosphorylase through AMP, Pi, and epinephrine (18) along with stimulation of phosphofructokinase (PFK) by AMP, Pi, and ADP (5). Therefore, each of these blood metabolites provides information about different, but related, causes of muscle fatigue.

In conclusion, differences of metabolite accumulation in the blood account for diverse energy system activity from the manipulation of resistance exercise variables. Levels of NH4 and La in the blood caused by resistance exercise are dependent on rest time between sets, time under tension, number of sets, number of repetitions, load, and work volume. Blood NH4 and La levels represent a detriment in muscle contractile properties and reduced capacity for ATP resynthesis. There is not a strong correlation between blood NH4 and La levels; however, they both increase in a similar manner as repetition numbers increase. Our study ratifies that NH4 and La are proficient markers of PCr depletion and glycogen utilization, allowing them to be useful indicators of muscle fatigue and metabolic load caused by resistance exercise.

Practical Applications

Knowledge of metabolic response to different strength training protocols will assist strength and conditioning professionals in understanding recovery needs. Ammonium and La are markers of metabolic load and resulting muscle fatigue; however, the information they provide is quite different. The presence of elevated NH4 reflects decreased energy levels in the muscle, whereas La represents muscle glycogen depletion and an indirect estimate of muscle pH. Loss of PCr denotes a short term, whereas a lower TAN pool represents a longer term inability to produce ATP rapidly by the phosphagen energy system, indicating muscle fatigue. Greater glycogen depletion in a muscle group during ME type training vs. volume equated HYP or STR training will require longer recovery times between training sessions, whereas a transient decrease in pH may further explain a metabolic mechanism for muscle fatigue. Therefore, strength and conditioning coaches using higher repetition, lower load protocols should be cognizant of these differences in muscle energy sources with short rest periods between sets.

If the purpose is to challenge muscle metabolism through resistance training, the results of this study indicate that high repetition, low-load protocols with short recovery time (approximately 45 seconds) provide high stress on muscle metabolism. The shorter rest between sets does not allow significant PCr recovery, stimulates the loss of TAN, and increases muscle glycogen depletion leading to early muscle fatigue. However, the short recovery time between sets should be avoided if the high repetition, low-load protocols are used by the strength and conditioning coach to provide a low-intensity recovery day. In this instance, increasing time between sets may be required to allow PCr recovery and avoid the cascade of events that stems from depletion of PCr in the active muscles. Because La levels also indicate high metabolic stress related to muscle glycogen depletion, but not PCr depletion, the usefulness of both NH4 and La gives more specific metabolic information to the coach and researcher about the likely sources of metabolic muscle fatigue. Because PCr can be restored in a few minutes, but muscle glycogen levels require much longer to be resynthesized, manipulation of rest between sets is indicated knowing the likely primary source of fatigue. Monitoring NH4 and La levels gives the coach and researcher insights to the metabolic state and fatigue of their athletes' muscles allowing him/her to plan superior individual workouts and to determine future directions for the athlete.


Full funding was provided by the Graduate Student Research, Service, and Education Leadership Grant Program at the University of Wisconsin-La Crosse, LA Crosse, WI. We would like to thank Christopher Dodge for his help with laboratory procedures, the provision of laboratory workspace, and providing the chemicals necessary to analyze plasma NH4 and blood La concentrations.


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purine nucleotide cycle; phosphocreatine; muscular endurance

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