The sit-and-reach test (SRT) has been included in standard fitness test batteries for decades, but empirical evidence of actual muscle activity has been lacking. Furthermore, the positioning of the ankle joint during the execution of this test has received relatively scant attention. Therefore, the purpose of this investigation was to compare surface electromyographic (sEMG) activity of selected lower extremity and back musculature and examine the impact of ankle positioning during the standard SRT and the modified sit-and-reach test (MSRT). Seven male and 7 female subjects performed 3 trials of the SRT and MSRT, each in a dorsiflexed and plantar flexed ankle position. During all trials, muscle activity (sEMG) was measured from the right semimembranous (SM), erector spinae (ES), and gastrocnemius (G). Mean sEMG data from each muscle (SM, ES, and G) were normalized by being expressed as a percent contribution to the total electrical activity (100%). Surface electromyographic activity data were also used to determine muscle activation ratios (e.g., SM to ES). Results revealed significantly higher flexibility scores during the plantar flexion condition for both test modalities. The SM exhibited the greatest percent contribution to total sEMG activity within all testing conditions. The SM to G and SM to ES muscle activation ratios were significantly greater than their inverse counterparts within all 4 testing conditions. Based on the 2 sEMG analysis techniques, the SM seemed to exhibit the greatest muscle activity. This investigation provides direct evidence of sEMG muscle activity during the SRT and MSRT, further confirming these tests to be a valid measure of hamstring flexibility.