1. The main purpose of this study was to evaluate non-invasively with magnetic resonance spectroscopy (1H-MRS) changes in the concentrations of intracellular (IT) and extracellular (between muscle fibres) triglycerides (ET) in skeletal muscles of trained males (age range: 24-38 years) during two standard exercise protocols of alternating velocities. 2. Protocol 1 consisted of locomotion in a shuttle manner between two lines 30 m apart at four different velocities (1, 2, 3, and 4 m s-1) which were alternated every minute in a standard routine for 90 min, whereas Protocol 2 included locomotion between two lines 20 m apart at only three velocities (2, 2.7 and 4 m s-1) until volitional exhaustion. The heart rate during both protocols fluctuated between 140 and 200 beats min-1. 3. Using pre-exercise muscle water to quantify individual total creatine (TCr) that was utilized as an internal standard and assuming that TCr does not change during exercise, subjects' mean IT and ET concentrations in soleus (Sol) muscle before Protocol 1 (n = 8) were 45.8 +/- 4.8 mmol (kg dry weight)-1 (mean +/- S.E.M.) and 93.1 +/- 14.1 mmol (kg dry weight)-1, respectively. After the exercise, the concentrations of IT and ET were not significantly different from the values at rest. Before Protocol 2 (n = 4), IT concentrations in Sol, gastrocnemius (Gast) and tibialis (Tib) muscles were 46.4 +/- 13.6, 35.0 +/- 12.1 and 23.1 +/- 4.8 mmol (kg dry weight)-1, respectively, and were not affected by the exhaustive exercise. The ET concentrations in Sol, Gast and Tib were 136.4 +/- 38.1, 175.3 +/- 86.5 and 79.3 +/- 20.0 mmol (kg dry weight)-1 respectively, and they did not change significantly after exhaustion. 4. The study showed that levels of IT and ET were not affected by alternating intensity exercise to fatigue. This suggests that IT and ET in human Sol, Gast and Tib muscles do not contribute significantly to the energy turnover during this type of exercise. Energy for this type of muscle contraction may arise primarily from muscle phosphocreatine (PCr) and glycogen breakdown, circulating glucose and fatty acids from triglycerides other than those encountered within and between muscle cells.