We investigated longitudinally the effect of protease inhibitors (PI) on insulin sensitivity, glycemia, and serum lipids in HIV-infected patients. Ninety-one consecutive patients treated with PI for at least 12 months were included in this study. Fasting glycemia, lipid profile, insulinemia, CD4 T lymphocytes, and plasma HIV-1 RNA were performed at baseline and on PI therapy. Insulin sensitivity and insulin secretion were measured by the homeostasis model assessment (HOMA MODEL) using the fasting glucose and insulin concentrations. Triglycerides (+ 0.34 mmol/l, SD = 1.07, p = 0.001) and cholesterol (+ 1.07 mmol/l, SD = 1.21, p= 0.001) significantly increased on PI therapy. Fasting glycemia, insulin sensitivity, and insulin secretion were not modified after PI therapy. PI therapy significantly increased body mass index (0.35 kg/m2, p < 0.05). Serum lipid changes correlated with changes in the CD4+ cell count. Lipodystrophy was observed in 40.6% of patients treated with PI. Our longitudinal study found that PI therapy had no major impact on fasting glycemia, insulin sensitivity, and insulin secretion. These findings are not consistent with previous cross-sectional studies, which did not include baseline measurements before PI initiation. However, we observed a similar profile of lipid changes induced by PI therapy. These results suggest that PI could be responsible for the development of hypertriglyceridemia by a mechanism independent of insulin resistance which remains to be elucidated.