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Summary Background Recommendations to use other criteria than N-balance for defining protein requirements have been proposed. However, little evidence to support other measures such as physiological functions is available. Objective To investigate the effects of a usual (UP) versus a high protein (HP) diet on muscle function, cognitive function, quality of life and biochemical regulators of protein metabolism. Design A randomised intervention study was conducted with 23 healthy males (aged 19–31 yrs). All subjects consumed a Usual Protein (UP) diet (1.5 g protein/kg BW) for a 1-wk run-in period before the intervention period where they were assigned to either a UP or a High Protein (HP) diet (3.0 g protein/kg BW) for 3-wks with controlled intake of food and beverages. Blood and urine samples were taken along with measurements of physiological functions at baseline and at the end of the intervention period. Results The HP group improved their reaction time significantly compared with the UP group. Branched chain amino acids and phenylalanine in plasma were significantly increased following the HP diet, which may explain the improved reaction time. Conclusion Healthy young males fed a HP diet improved reaction time. No adverse effects of the HP diet were observed. This trial was registered at www.clinicaltrials.govas NCT00621231.