The aim of this study was twofold: (i) to describe the criterion-related validity of the sit-and-reach test (SRT) using a hand-held inclinometer when assessing hamstring muscle length (HML) when HML is recorded in degrees of hip joint angle (HJA); and (ii) to describe the effect of gender and age on HML in healthy adults during the performance of a SRT. We examined 212 healthy subjects (106 men and 106 women) whose ages ranged from 20 to 79 years. The Pearson-product moment correlation coefficient (r) described the relationship between HJA at the end-point of the SRT and the criterion, supine passive straight-leg raise (PSLR). We conducted a 6 x 2 analysis of variance, where age was stratified on 6 levels of 10-year increments (20-29, 30-39, 40-49, 50-59, 60-69, and 70-79 years of age) and gender was stratified on 2 levels (men and women). There was a statistically significant correlation (r = 0.59, P < 0.01) between performance on the SRT as measured by HJA and the supine PSLR, but the SRT only accounted for 35% of the variability in the PSLR. SRT performance in men (mean +/- SD, 80 +/- 9 degrees) was significantly less (P < 0.001) than in women (mean +/- SD, 92 +/- 10 degrees). Subjects in the 60- to 69- and 70- to 79-year age groups had significantly less (P < 0.05) HJA than those in the 20- to 29-, 30- to 39-, and 40- to 49-year age groups. Using an inclinometer to measure HJA during the SRT is not a valid method for assessing HML in men and women who can independently assume a long-sitting position on a hard surface. Clinicians should recognize there are differences in HML between men and women, and that men and women between 20 to 49 years of age have more HML than their counterparts between ages 60 to 79 years.